Induction of Pluripotent Stem Cells from Adult Human
Fibroblasts by Defined Factors
Takahashi, Kazutoshi; Tanabe, Koji; Ohnuki, Mari; Narita,
Megumi; Ichisaka, Tomoko; Tomoda, Kiichiro; Yamanaka,
CitationCell (2007), 131(5): 861-872
Issue Date 2007-11-30
RightCopyright © 2007 Elsevier B.V. All rights reserved.
KURENAI : Kyoto University Research Information Repository
Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined
Kazutoshi Takahashi1, Koji Tanabe1, Mari Ohnuki1, Megumi Narita1,2, Tomoko Ichisaka1,2,
Kiichiro Tomoda3and Shinya Yamanaka1–4*
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
3Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158
4Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan
* [D] Manuscript
Successful reprogramming of differentiated human somatic cells into a pluripotent state
would allow creation of patient- and disease-specific stem cells. We previously reported
generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from
mouse somatic cells by transduction of four defined transcription factors. Here, we
demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same
four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human
embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression,
epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore,
these cells could differentiate into cell types of the three germ layers in vitro and in
teratomas. These findings demonstrate that iPS cells can be generated from adult human
Embryonic stem (ES) cells, derived from the inner cell mass of mammalian blastocysts,
have the ability to grow indefinitely while maintaining pluripotency(Evans and Kaufman,
1981; Martin, 1981). These properties have led to expectations that human ES cells might
be useful to understand disease mechanisms, to screen effective and safe drugs, and to treat
patients of various diseases and injuries, such as juvenile diabetes and spinal cord
injury(Thomson et al., 1998). Use of human embryos, however, faces ethical controversies
that hinder the applications of human ES cells. In addition, it is difficult to generate patient-
or disease-specific ES cells, which are required for their effective application. One way to
circumvent these issues is to induce pluripotent status in somatic cells by direct
We showed that induced pluripotent stem (iPS) cells can be generated from mouse
embryonic fibroblasts (MEF) and adult mouse tail-tip fibroblasts by the retrovirus-mediated
transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4(Takahashi
and Yamanaka, 2006). Mouse iPS cells are indistinguishable from ES cells in morphology,
proliferation, gene expression, and teratoma formation. Furthermore, when transplanted
into blastocysts, mouse iPS cells can give rise to adult chimeras, which are competent for
germline transmission(Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007). These
results are proof-of-principle that pluripotent stem cells can be generated from somatic cells
by the combination of a small number of factors.
In the current study, we sought to generate iPS cells from adult human somatic
cells by optimizing retroviral transduction in human fibroblasts and subsequent culture
conditions. These efforts have enabled us to generate iPS cells from adult human dermal
fibroblasts and other human somatic cells, which are comparable to human ES cells in their
differentiation potential in vitro and in teratomas.
Optimization of Retroviral Transduction for Generating Human iPS Cells
Induction of iPS cells from mouse fibroblasts requires retroviruses with high transduction
efficiencies(Takahashi and Yamanaka, 2006). We, therefore, optimized transduction
methods in adult human dermal fibroblasts (HDF). We first introduced green fluorescent
protein (GFP) into adult HDF with amphotropic retrovirus produced in PLAT-A packaging
cells. As a control, we introduced GFP to mouse embryonic fibroblasts (MEF) with
ecotropic retrovirus produced in PLAT-E packaging cells(Morita et al., 2000). In MEF,
more than 80% of cells expressed GFP (S-Figure 1). In contrast, less that 20% of HDF
expressed GFP with significantly lower intensity than in MEF. To improve the transduction
efficiency, we introduced the mouse receptor for retroviruses, Slc7a1(Verrey et al., 2004)
(also known as mCAT1), into HDF with lentivirus. We then introduced GFP to HDF-Slc7a1
with ecotropic retrovirus. This strategy yielded a transduction efficiency of 60%, with a
similar intensity to that in MEF.
Generation of iPS Cells from Adult HDF
The protocol for human iPS cell induction is summarized in Figure 1A. We introduced the
retroviruses containing human Oct3/4, Sox2, Klf4 and c-Myc into HDF-Slc7a1 (Figure 1B,
8 x 105 cells per 100 mm dish). The HDF derived from facial dermis of 36-year-old
Caucasian female. Six days after transduction, the cells were harvested by trypsinization
and plated onto mitomycin C-treated SNL feeder cells(McMahon and Bradley, 1990) at 5 x
104 or 5 x 105 cells per 100-mm dish. The next day, the medium (DMEM containing 10%
FBS) was replaced with a medium for primate ES cell culture supplemented with 4 ng/ml
basic fibroblast growth factor (bFGF).
Approximately two weeks later, some granulated colonies appeared that were not
similar to hES cells in morphology (Figure 1C). Around day 25, we observed distinct types
of colonies that were flat and resembled hES cell colonies (Figure 1D). From 5 x 104
fibroblasts, we observed ~10 hES cell–like colonies and ~100 granulated colonies (7/122,
8/84, 8/171, 5/73, 6/122 and 11/213 in six independent experiments, summarized in
Supplemental Table 1). At day 30, we picked hES cell–like colonies and mechanically
disaggregated them into small clumps without enzymatic digestion. When starting with 5 x
105 fibroblasts, the dish was nearly covered with more than 300 granulated colonies. We
occasionally observed some hES cell–like colonies in between the granulated cells, but it
was difficult to isolate hES cell–like colonies because of the high density of granulated
cells. The nature of the non- hES-like cells remains to be determined.
The hES-like cells expanded on SNL feeder cells with the primate ES cell medium
containing bFGF. They formed tightly packed and flat colonies (Figure 1E). Each cell
exhibited morphology similar to that of human ES cells, characterized by large nuclei and
scant cytoplasm (Figure 1F). As is the case with hES cells, we occasionally observed
spontaneous differentiation in the center of the colony (Fig. 1G).
These cells also showed similarity to hES cells in feeder dependency (S-Figure 2).
They did not attach to gelatin-coated tissue-culture plates. By contrast, they maintained an
undifferentiated state on Matrigel-coated plates in MEF-conditioned primate ES cell
medium, but not in non-conditioned medium.
Since these cells were similar to hES cells in morphology and other aspects noted
above, we will refer to the selected cells after transduction of HDF as human iPS cells, as
we describe the molecular and functional evidence for this claim. Human iPS cells clones
established in this study are summarized in S-Table 2.
Human iPS Cells Express hES Markers
In general, except for a few cells at the edge of the colonies, human iPS cells did not
express stage-specific embryonic antigen (SSEA)-1 (Figure 1H). In contrast, they expressed
hES cell–specific surface antigens(Adewumi et al., 2007), including SSEA-3, SSEA-4,
tumor-related antigen (TRA) -1-60, TRA-1-81 and TRA-2-49/6E (alkaline phosphatase),
and NANOG protein (Fig. 1I~N).
RT-PCR showed human iPS cells expressed many undifferentiated ES cell marker
genes(Adewumi et al., 2007), such as OCT3/4, SOX2, NANOG, growth and differentiation
factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4),
embryonic cell–specific gene 1 (ESG1), developmental pluripotency–associated 2 (DPPA2),
DPPA4 and telomerase reverse transcriptase (hTERT) at levels equivalent to or higher than
those in the hES cell line H9 and the human embryonic carcinoma cell line, NTERA-2
(Figure 2A). By western blotting, proteins levels of OCT3/4, SOX2, NANOG, SALL4,
E-CADHERIN, and hTERT were similar in human iPS cells and hES cells (Figure 2B).
Although the expression levels of Klf4 and c-Myc increased more than five fold in HDF
after the retroviral transduction (not shown), their expression levels in human iPS cells
were comparable to those in HDF (Figure 2A & B), indicating retroviral silencing. RT-PCR
using primers specific for retroviral transcripts confirmed efficient silencing of all the four
retroviruses (Figure 2C). DNA microarray analyses showed that the global gene expression
patters are similar, but not identical, between human iPS cells and hES cells (Figure 2D).
Among 32266 gene analyzed, 5107 genes showed more than 5-fold difference in
expression between HDF and human iPS cells, whereas 1267 genes between human iPS
cells and hES cells (S-Table 3 & 4).
Promoters of ES Cell–Specific Genes Are Active in Human iPS Cells
Bisulfite genomic sequencing analyses evaluating the methylation statuses of cytosine
guanine dinucleotides (CpG) in the promoter regions of pluripotent-associated genes, such
as OCT3/4, REX1 and NANOG, revealed that they were highly unmethylated, whereas the
CpG dinucleotides of the regions were highly methylated in parental HDFs (Figure 3A).
These findings indicate that these promoters are active in human iPS cells.
Luciferase reporter assays also showed that human OCT3/4 and REX1 promoters
had high levels of transcriptional activity in human iPS cells and EC cells (NTERA-2), but
not in HDF. The promoter activities of ubiquitously expressed genes, such as human RNA
polymerase II (PolII), showed similar activities in both human iPS cells and HDF (Figure
We also performed chromatin immunoprecipitation to analyze the histone
modification status in human iPS cells (Figure 3C). We found that histone H3 lysine 4 was
methylated whereas H3 lysine 27 was demethylated in the promoter regions of Oct3/4,
Sox2, and Nanog in human iPS cells. We also found that human iPS cells showed the
bivalent patterns of development-associated genes, such as Gata6, Msx2, Pax6, and Hand1.
These histone modification statuses are characteristic of hES cells (Pan et al., 2007).
High Telomerase Activity and Exponential Growth of Human iPS Cells
As predicted from the high expression levels of hTERT, human iPS cells showed high
telomerase activity (Figure 4A). They proliferated exponentially for as least 4 months
(Figure 4B). The calculated population doubling time of human iPS cells were 46.9 ± 12.4
(clone 201B2), 47.8 ± 6.6 (201B6) and 43.2 ± 11.5 (201B7) hours. These times are
equivalent to the reported doubling time of hES cells(Cowan et al., 2004).
Embryoid Body–Mediated Differentiation of Human iPS Cells
To determine the differentiation ability of human iPS cells in vitro, we used floating
cultivation to form embryoid bodies (EBs)(Itskovitz-Eldor et al., 2000). After 8 days in
suspension culture, iPS cells formed ball-shaped structures (Figure 5A). We transferred
these embryoid body–like structures to gelatin-coated plates and continued cultivation for
another 8 days. Attached cells showed various types of morphologies, such as those
resembling neuronal cells, cobblestone-like cells, and epithelial cells (Figure 5B–E).
Immunocytochemistry detected cells positive for βIII-tubulin (a marker of ectoderm), glial
fibrillary acidic protein (GFAP, ectoderm), α-smooth muscle actin (α-SMA, mesoderm),
desmin (mesoderm), α-fetoprotein (AFP, endoderm), and vimentin (mesoderm and parietal
endoderm) (Figure 5F–K). RT-PCR confirmed that these differentiated cells expressed
forkhead box A2 (FOXA2, a marker of endoderm), AFP (endoderm), cytokeratin 8 and 18
(endoderm), SRY-box containing gene 17 (SOX17, endoderm), BRACHYURY (mesoderm),
Msh homeobox 1 (MSX1, mesoderm), microtubule-associated protein 2 (MAP2, ectoderm)
and paired box 6 (PAX6, ectoderm) (Figure 5L). In contrast, expression of OCT3/4, SOX2
and NANOG was markedly decreased. These data demonstrated that iPS cells could
differentiate into three germ layers in vitro.
Directed Differentiation of Human iPS Cells into Neural Cells
We next examined whether lineage-directed differentiation of human iPS cells could be
induced by reported methods for hES cells. We seeded human iPS cells on PA6 feeder layer
and maintained them under differentiation conditions for two weeks(Kawasaki et al., 2000).
Cells spread drastically, and some neuronal structures were observed (Figure 6A).
Immunocytochemistry detected cells positive for tyrosine hydroxylase and βIII tubulin in
the culture (Figure 6B). PCR analysis revealed expression of dopaminergic neuron markers,
such as aromatic-L–amino acid decarboxylase (AADC), member 3 (DAT), choline
acetyltransferase (ChAT), and LIM homeobox transcription factor 1 beta (LMX1B), as well
as another neuron marker, MAP2 (Figure 6C). In contrast, GFAP expression was not
induced with this system. On the other hand, the expression of OCT3/4 and NANOG
decreased markedly whereas Sox decreased only slightly (Figure 6C). These data
demonstrated that iPS cells could differentiate into neuronal cells, including dopaminergic
neurons, by co-culture with PA6 cells.
Directed Differentiation of Human iPS Cells into Cardiac Cells
We next examined directed cardiac differentiation of human iPS cells with the recently
reported protocol which utilizes activin A and bone morphogenetic protein (BMP)
4(Laflamme et al., 2007). Twelve days after the induction of differentiation, clumps of cells
started beating (Figure 6D, Supplemental movie). RT-PCR showed that these cells
expressed cardiomyocyte markers, such as troponin T type 2 cardiac (TnTc), myocyte
enhancer factor 2C (MEF2C), myosin, light polypeptide 7, regulatory (MYL2A), myosin,
heavy polypeptide 7, cardiac muscle, beta (MYHCB), and NK2 transcription factor related,
locus 5 (NKX2.5) (Figure 6E). In contrast, the expression of Oct3/4, Sox2, and Nanog
markedly decreased. Thus, human iPS cells can differentiate into cardiac myocytes in vitro.
Teratoma Formation from Human iPS Cells
To test pluripotency in vivo, we transplanted human iPS cells (clone 201B7)
subcutaneously into dorsal flanks of immunodeficient (SCID) mice. Nine weeks after
injection, we observed tumor formation. Histological examination showed that the tumor
contained various tissues (Figure 7), including gut-like epithelial tissues (endoderm),
striated muscle (mesoderm), cartilage (mesoderm), neural tissues (ectoderm), and
keratin-containing epidermal tissues (ectoderm).
Human iPS Cells Are Derived from HDF, not Cross-Contamination
PCR of genomic DNA of human iPS cells showed that all clones have integration of all the
four retroviruses (S-Figure 3A). Southern blot analysis with a c-Myc cDNA probe revealed
that each clone had a unique pattern of retroviral integration sites (S-Figure 3B). In addition,
the patterns of 16 short tandem repeats were completely matched between human iPS
clones and parental HDF (S-Table 5). These patterns differed from any established hES cell
lines reported on National Institutes of Health website
(http://stemcells.nih.gov/research/nihresearch/scunit/genotyping.htm). In addition,
chromosomal G-band analyses showed that human iPS cells had a normal karyotype of
46XX (not shown). Thus, human iPS clones were derived from HDF and were not a result
of cross-contamination. Whether generation of human iPS cells depends on minor genetic
or epigenetic modification awaits further investigation.
Generation of iPS Cells from Other Human Somatic Cells
In addition to HDF, we used primary human fibroblast-like synoviocytes (HFLS) from
synovial tissue of 69-year-old Caucasian male and BJ cells, a cell line established from
neonate fibroblasts (S-Table 1 & 2). From HFLS (5 x 104 cells per 100-mm dish), we
obtained more than 600 hundred granulated colonies and 17 hES cell–like colonies
(S-Table 1). We picked six colonies, of which only two were expandable as iPS cells
(S-figure 4). Dishes seeded with 5 x 105 HFLS were covered with granulated cells, and no
hES cell-like colonies were distinguishable. In contrast, we obtained 7–8 and ~100 hES
cell-like colonies from 5 x 104 and 5 x 105 BJ cells, respectively, with only a few granulated
colonies (S-Table 1). We picked six hES cell–like colonies and generated iPS cells from
five colonies (S-Figure 4). Human iPS cells derived from HFLS and BJ expressed hES cell
marker genes at levels similar to or higher than those in hES cells (S-Figure 5). They
differentiated into all three germ layers through EBs (S-Figure 6). STR analyses confirmed
that iPS-HFLS cells and iPS-BJ cells were derived from HFLS and BJ fibroblasts,
respectively (S-Table 6 & 7).
In this study, we showed that iPS cells can be generated from adult HDF and other somatic
cells by retroviral transduction of the same four transcription factors with mouse iPS cells,
namely Oct3/4, Sox2, Klf4, and c-Myc. The established human iPS cells are similar to hES
cells in many aspects, including morphology, proliferation, feeder dependence, surface
markers, gene expression, promoter activities, telomerase activities, in vitro differentiation,
and teratoma formation. The four retroviruses are strongly silenced in human iPS cells,
indicating that these cells are efficiently reprogrammed and do not depend on continuous
expression of the transgenes for self-renewal.
hES cells are different from mouse counterparts in many respects(Rao, 2004). hES
cell colonies are flatter and do not override each other. hES cells depend on bFGF for
self-renewal(Amit et al., 2000), whereas mouse ES cells depend on the LIF/Stat3
pathway(Matsuda et al., 1999; Niwa et al., 1998). BMP induces differentiation in hES
cells(Xu et al., 2005) but is involved in self-renewal of mouse ES cells(Ying et al., 2003).
Despite these differences, our data show that the same four transcription factors induce iPS
cells in both human and mouse. The four factors, however, could not induce human iPS
cells when fibroblasts were kept under the culture condition for mouse ES cells after
retroviral transduction (data not shown). These data suggest that the fundamental
transcriptional network governing pluripotency is common in human and mice, but
extrinsic factors and signals maintaining pluripotency are unique for each species.
Deciphering of the mechanism by which the four factors induce pluripotency in
somatic cells remains elusive. The function of Oct3/4 and Sox2 as core transcription factors
to determine pluripotency is well documented(Boyer et al., 2005; Loh et al., 2006; Wang et
al., 2006). They synergistically up-regulate “stemness” genes, while suppressing
differentiation-associated genes in both mouse and human ES cells. However, they cannot
bind their targets genes in differentiated cells, because of other inhibitory mechanisms,
including DNA methylation and histone modifications. We speculate that c-Myc and Klf4
modifies chromatin structure so that Oct3/4 and Sox2 can bind to their targets(Yamanaka,
2007). Notably, Klf4 interacts with p300 histone acetyltransferase and regulates gene
transcription by modulating histone acetylation(Evans et al., 2007).
The negative role of c-Myc in the self-renewal of hES cells was recently
reported(Sumi et al., 2007). They showed that forced-expression of c-Myc induced
differentiation and apoptosis of human ES cells. This is great contrast to the positive role of
c-Myc in mouse ES cells(Cartwright et al., 2005). During iPS cell generation, transgenes
derived from retroviruses are silenced when the transduced fibroblasts acquire ES-like state.
The role of c-Myc in establishing iPS cells may be as a booster of reprogramming, rather
than a controller of maintenance of pluripotency.
We found that each iPS clone contained 3–6 retroviral integrations for each factor.
Thus, each clone had more than 20 retroviral integration sites in total, which may increase
the risk of tumorigenesis. In the case of mouse iPS cells, ~20% of mice derived from iPS
cells developed tumors, which were attributable, at least in part, to reactivation of the
c-Myc retrovirus (Okita et al., 2007). This issue must be overcome to use iPS cells in
human therapies. We have recently found that iPS cells can be generated without Myc
retroviruses, albeit with lower efficiency (Nakagawa, M., Koyanagi, M., and Yamanaka, S.,
submitted). Non-retroviral methods to introduce the remaining three factors, such as
adenoviruses or cell-permeable recombinant proteins, should be examined in future studies.
Alternatively, one might be able to identify small molecules that can induce iPS cells,
without gene transfer.
As is the case with mouse iPS cells, only a small portion of human fibroblasts that
had been transduced with the four retroviruses acquired iPS cell identity. We obtained ~10
iPS cells colonies from 5 x 104 transduced HDF. From a practical point of view, this
efficiency is sufficiently high since multiple iPS cell clones can be obtained from a single
experiment. From a scientific point of view, however, the low efficiency raises several
possibilities. First, the origin of iPS cells may be undifferentiated stem or progenitor cells
co-existing in fibroblast culture. Another possibility is that retroviral integration into some
specific loci may be required for iPS cell induction. Finally, minor genetic alterations,
which could not be detected by karyotype analyses, or epigenetic alterations are required
for iPS cell induction. These issues need to be elucidated in future studies.
Our study has opened an avenue to generate patient- and disease-specific
pluripotent stem cells. Even with the presence of retroviral integration, human iPS cells are
useful for understanding disease mechanisms, drug screening, and toxicology. For example,
hepatocytes derived from iPS cells with various genetic and disease backgrounds can be
utilized in predicting liver toxicity of drug candidates. Once the safety issue is overcome,
human iPS cells should also be applicable in regenerative medicine. Human iPS cells,
however, are not identical to hES cells: DNA microarray analyses detected differences
between the two pluripotent stem cell lines. Further studies are essential to determine
whether human iPS cells can replace hES in medical applications.
HDF from facial dermis of 36-year-old Caucasian female and HFLS from synovial tissue of
69-year-old Caucasian male were purchased from Cell Applications, Inc. When received,
the population doubling was less than 16 in HDF and 5 in HFLS. We used these cells for
the induction of iPS cells within six and four passages after the receipt. BJ fibroblasts from
neonatal foreskin and NTERA-2 clone D1 human embryonic carcinoma cells were obtained
from American Type Culture Collection. Human fibroblasts, NTERA-2, PLAT-E and
PLAT-A cells were maintained in Dulbecco’s modified eagle medium (DMEM, Nacalai
Tesque, Japan) containing 10% fetal bovine serum (FBS, Japan Serum) and 0.5% penicillin
and streptomycin (Invitrogen). 293FT cells were maintained in DMEM containing 10%
FBS, 2 mM L-glutamine (Invitrogen), 1 x 10-4 M nonessential amino acids (Invitrogen), 1
mM sodium pyruvate (Sigma) and 0.5% penicillin and streptomycin. PA6 stroma cells
(RIKEN Bioresource Center, Japan) were maintained in α-MEM containing 10% FBS and
0.5% penicillin and streptomycin. iPS cells were generated and maintained in Primate ES
medium (ReproCELL, Japan) supplemented with 4 ng/ml recombinant human basic
fibroblast growth factor (bFGF, WAKO, Japan). For passaging, human iPS cells were
washed once with PBS, and then incubated with DMEM/F12 containing 1 mg/ml
collagenase IV (Invitrogen) at 37oC. When colonies at the edge of the dish started
dissociating from the bottom, DMEF/F12/collangenase was removed and washed with
primate ES cell medium. Cells were scraped and collected into 15-ml conical tube. An
appropriate volume of the medium was added, and the contents were transferred to a new
dish on SNL feeder cells. The split ratio was routinely 1:3. For feeder-free culture of iPS
cells, the plate was coated with 0.3 mg/ml Matrigel (Growth factor reduced, BD
Biosciences) at 4oC overnight. The plate was warmed to room temperature before use.
Unbound Matrigel was aspirated off and washed out with DMEM/F12. iPS cells were
seeded on Matrigel-coated plate in MEF-conditioned or non-conditioned primate ES cell
medium, both supplemented with 4 ng/ml bFGF. The medium was changed daily. For
preparation of MEF-conditioned medium, MEFs derived from embryonic day 13.5 embryo
pool of ICR mice were plated at 1 x 106 cells per 100-mm dish and incubated overnight.
Next day, the cells were washed once with PBS, and cultured in 10 ml of primate ES cell
medium. After twenty-four hour incubation, the supernatant of MEF culture was collected,
filtered through a 0.22-μm pore-size filter, and stored at -20oC until use.
The open reading frame of human OCT3/4 was amplified by RT-PCR and cloned into
pCR2.1-TOPO. An EcoRI fragment of pCR2.1-hOCT3/4 was introduced into the EcoRI
site of pMXs retroviral vector. To discriminate each experiment, we introduced a 20-bp
random sequence, which we designated N20 barcode, into the NotI/SalI site of Oct3/4
expression vector. We used a unique barcode sequence in each experiment to avoid
inter-experimental contamination. The open reading frames of human SOX2, KLF4 and
c-MYC were also amplified by RT-PCR and subcloned into pENTR-D-TOPO (Invitrogen).
All of the genes subcloned into pENTR-D-TOPO were transferred to pMXs by using the
Gateway cloning system (Invitrogen), according to the manufacturer’s instructions. Mouse
Slc7a1 ORF was also amplified, subcloned into pENTR-D-TOPO, and transferred to
pLenti6/UbC/V5-DEST (Invitrogen) by the Gateway system. The regulatory regions of the
human OCT3/4 gene and the REX1 gene were amplified by PCR and subcloned into
pCRXL-TOPO (Invitrogen). For phOCT4-Luc and phREX1-Luc, the fragments were
removed by KpnI/BglII digestion from pCRXL vector and subcloned into the KpnI/BglII
site of pGV-BM2. For pPolII-Luc, an AatII (blunted)/NheI fragment of pQBI-polII was
inserted into the KpnI (blunted)/NheI site of pGV-BM2. All of the fragments were verified
by sequencing. Primer sequences are shown in S-Table 8.
Lentivirus Production and Infection
293FT cells (Invitrogen) were plated at 6 x 106 cells per 100-mm dish, and incubated
overnight. Cells were transfected with 3 μg of pLenti6/UbC-Slc7a1 along with 9 μg of
Virapower packaging mix by Lipofectamine 2000 (Invitrogen), according to the
manufacturer’s instructions. Forty-eight h after transfection, the supernatant of transfectant
was collected and filtered through a 0.45-μm pore-size cellulose acetate filter (Whatman).
Human fibroblasts were seeded at 8 x 105 cells per 100-mm dish 1 day before transduction.
The medium was replaced with virus-containing supernatant supplemented with 4 μg/ml
polybrene (Nacalai Tesque), and incubated for 24 h.
Retroviral Infection and iPS Cell Generation
PLAT-E packaging cells were plated at 8 x 106 cells per 100-mm dish and incubated
overnight. Next day, the cells were transfected with pMXs vectors with Fugene 6
transfection reagent (Roche). Twenty-four h after transfection, the medium was collected as
the first virus-containing supernatant and replaced with a new medium, which was
collected after 24 h as the second virus-containing supernatant. Human fibroblasts
expressing mouse Slc7a1 gene were seeded at 8 x 105 cells per 100-mm dish 1 day before
transduction. The virus-containing supernatants were filtered through a 0.45-μm pore-size
filter, and supplemented with 4 μg/ml polybrene. Equal amounts of supernatants containing
each of the four retroviruses were mixed, transferred to the fibroblast dish, and incubated
overnight. Twenty-four h after transduction, the virus-containing medium was replaced
with the second supernatant. Six days after transduction, fibroblasts were harvested by
trypsinization and re-plated at 5 x 104 cells per 100-mm dish on an SNL feeder layer. Next
day, the medium was replaced with hES medium supplemented with 4 ng/ml bFGF. The
medium was changed every other day. Thirty days after transduction, colonies were picked
up and transferred into 0.2 ml of hES cell medium. The colonies were mechanically
dissociated to small clamps by pipeting up and down. The cell suspension was transferred
on SNL feeder in 24-well plates. We defined this stage as passage 1.
RNA Isolation and Reverse Transcription
Total RNA was purified with Trizol reagent (Invitrogen) and treated with Turbo DNA-free
kit (Ambion) to remove genomic DNA contamination. One microgram of total RNA was
used for reverse transcription reaction with ReverTraAce-α (Toyobo, Japan) and dT20
primer, according to the manufacturer’s instructions. PCR was performed with ExTaq
(Takara, Japan). Quantitative PCR was performed with Platinum SYBR Green qPCR
Supermix UDG (Invitrogen) and analyzed with the 7300 real-time PCR system (Applied
Biosystems). Primer sequences are shown in S-Table 8.
Alkaline Phosphatase Staining and Immunocytochemistry
Alkaline phosphatase staining was performed using the Leukocyte Alkaline Phosphatase kit
(Sigma). For immunocytochemistry, cells were fixed with PBS containing 4%
paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were
treated with PBS containing 5% normal goat or donkey serum (Chemicon), 1% bovine
serum albumin (BSA, Nacalai tesque), and 0.1% TritonX-100 for 45 min at room
temperature. Primary antibodies included SSEA1 (1:100, Developmental Studies
Hybridoma Bank), SSEA3 (1:10, a kind gift from Dr. Peter W. Andrews), SSEA4 (1:100,
Developmental Studies Hybridoma Bank), TRA-2-49/6E (1:20, Developmental Studies
Hybridoma Bank), TRA-1-60 (1:50, a kind gift from Dr. Peter W. Andrews), TRA-1-81
(1:50, a kind gift from Dr. Peter W. Andrews), Nanog (1:20, AF1997, R&D Systems),
βIII-tubulin (1:100, CB412, Chemicon), glial fibrillary acidic protein (1:500, Z0334,
DAKO), α-smooth muscle actin (pre-diluted, N1584, DAKO), desmin (1:100, RB-9014,
Lab Vision), vimentin (1:100, SC-6260, Santa Cruz), α-fetoprotein (1:100, MAB1368,
R&D Systems), tyrosine hydroxylase (1:100, AB152, Chemicon). Secondary antibodies
used were cyanine 3 (Cy3) –conjugated goat anti-rat IgM (1:500, Jackson Immunoresearch),
Alexa546-conjugated goat anti-mouse IgM (1:500, Invitrogen), Alexa488-conjugated goat
anti-rabbit IgG (1:500, Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:500,
Invitrogen), Cy3-conjugated goat anti-mouse IgG (1:500, Chemicon), and
Alexa488-conjugated goat anti-mouse IgG (1:500, Invitrogen). Nucleuses were stained
with 1 μg/ml Hoechst 33342 (Invitrogen).
In Vitro Differentiation
For EB formation, human iPS cells were harvested by treating with collagenase IV. The
clumps of the cells were transferred to poly (2-hydroxyrthyl methacrylate)–coated dish in
DMEM/F12 containing 20% knockout serum replacement (KSR, Invitrogen), 2 mM
L-glutamine, 1 x 10-4 M non essential amino acids, 1 x 10-4 M 2-mercaptoethanol
(Invitrogen), and 0.5% penicillin and streptomycin. The medium was changed every other
day. After 8 days as a floating culture, EBs were transferred to gelatin-coated plate and
cultured in the same medium for another 8 days. Co-culture with PA6 was used for
differentiation into dopaminergic neurons. PA6 cells were plated on gelatin-coated 6-well
plates and incubated for 4 days to reach confluence. Small clumps of iPS cells were plated
on PA6-feeder layer in Glasgow minimum essential medium (Invitrogen) containing 10%
KSR (Invitrogen), 1 x 10-4 M nonessential amino acids, 1 x 10-4 M 2-mercaptoethanol
(Invitrogen), and 0.5% penicillin and streptomycin. For cardiomyocyte differentiation, iPS
cells were maintained on Matrigel-coated plate in MEF-CM supplemented with 4 ng/ml
bFGF for 6 days. The medium was then replaced with RPMI1640 (Invitrogen) plus B27
supplement (Invitrogen) medium (RPMI/B27), supplemented with 100 ng/ml human
recombinant activin A (R & D Systems) for 24 h, followed by 10 ng/ml human recombinant
bone morphologenic protein 4 (BMP4, R & D Systems) for 4 days. After cytokine
stimulation, the cells were maintained in RPMI/B27 without any cytokines. The medium
was changed every other day.
Genomic DNA (1 g) was treated with CpGenome DNA modification kit (Chemicon),
according to the manufacturer’s recommendations. Treated DNA was purified with
QIAquick column (QIAGEN). The promoter regions of the human Oct3/4, Nanog and
Rex1 genes were amplified by PCR. The PCR products were subcloned into pCR2.1-TOPO.
Ten clones of each sample were verified by sequencing with the M13 universal primer.
Primer sequences used for PCR amplification were provided in S-Table 8.
Each reporter plasmid (1 g) containing the firefly luciferase gene was introduced into
human iPS cells or HDF with 50 ng of pRL-TK (Promega). Forty-eight h after transfection,
the cells were lysed with 1 x passive lysis buffer (Promega) and incubated for 15 min at
room temperature. Luciferase activities were measured with a Dual-Luciferase reporter
assay system (Promega) and Centro LB 960 detection system (BERTHOLD), according to
the manufacturer’s protocol.
The cells were harvested by collagenase IV treatment, collected into tubes and centrifuged,
and the pellets were suspended in DMEM/F12. One quarter of the cells from a confluent
100-mm dish was injected subcutaneously to dorsal flank of a SCID mouse （CREA, Japan).
Nine weeks after injection, tumors were dissected, weighted and fixed with PBS containing
4% paraformaldehyde. Paraffin-embedded tissue was sliced and stained with hematoxylin
The cells at semi-confluent state were lysed with RIPA buffer (50 mM Tris-HCl, pH 8.0,
150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, and 0.1% SDS),
supplemented with protease inhibitor cocktail (Roche). The cell lysate of MEL-1 hES cell
line was purchased from Abcam. Cell lysates (20 g) were separated by electrophoresis on
8% or 12% SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride
membrane (Millipore). The blot was blocked with TBST (20 mM Tris-HCl, pH 7.6, 136
mM NaCl, and 0.1% Tween-20) containing 1% skim milk and then incubated with primary
antibody solution at 4oC overnight. After washing with TBST, the membrane was incubated
with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room
temperature. Signals were detected with Immobilon Western chemiluminescent HRP
substrate (Millipore) and LAS3000 imaging system (FUJIFILM, Japan). Antibodies used
for western blotting were anti-Oct3/4 (1:600, SC-5279, Santa Cruz), anti-Sox2 (1:2000,
AB5603, Chemicon), anti-Nanog (1:200, R&D Systems), anti-Klf4 (1:200, SC-20691,
Santa Cruz), anti-c-Myc (1:200, SC-764, Santa Cruz), anti-E-cadherin (1:1000, 610182,
BD Biosciences), anti-Dppa4 (1:500, ab31648, Abcam), anti-FoxD3 (1:200, AB5687,
Chemicon), anti-telomerase (1:1000, ab23699, Abcam), anti-Sall4 (1:400, ab29112,
Abcam), anti-LIN-28 (1:500, AF3757, R&D systems), anti-β-actin (1:5000, A5441, Sigma),
anti-mouse IgG-HRP (1:3000, #7076, Cell Signaling), anti-rabbit IgG (1:2000, #7074, Cell
Signaling), and anti-goat IgG-HRP (1:3000, SC-2056, Santa Cruz)
Genomic DNA (5 g) was digested with BglII, EcoRI and NcoI overnight. Digested DNA
fragments were separated on 0.8% agarose gel and transferred to a nylon membrane
(Amersham). The membrane was incubated with digoxigenin (DIG) -labeled DNA probe in
DIG Easy Hyb buffer (Roche) at 42oC overnight with constant agitation. After washing,
alkaline phosphatase-conjugated anti-DIG antibody (1:10000, Roche) was added to a
membrane. Signals were raised by CDP-star (Roche) and detected by LAS3000 imaging
Short Tandem Repeat Analysis and Karyotyping
The genomic DNA was used for PCR with Powerplex 16 system (Promega) and analyzed
by ABI PRISM 3100 Genetic analyzer and Gene Mapper v3.5 (Applied Biosystems).
Chromosomal G-band analyses were performed at the Nihon Gene Research Laboratories,
Detection of Telomerase Activity
Telomerase activity was detected with a TRAPEZE telomerase detection kit (Chemicon),
according to the manufacturer’s instructions. The samples were separated by TBE-based
10% acrylamide non-denaturing gel electrophoresis. The gel was stained with SYBR Gold
Chromatin immunoprecuipitation assay
Approximately 1 x 107 cells were cross-linked with 1% formaldehyde for 5 minutes at room temperature,
and quenched by addition of glycine. The cell lysate was sonicated to share chromatin-DNA complex.
Immunoprecipitation was performed with Dynabeads Protein G (Invitrogen) -linked anti-trimethyl Lys 4
histone H3 (07-473, Upstate), anti-trimethyl Lys 27 histone H3 (07-449, Upstate) or normal rabbit IgG
antibody. Eluates were used for quantitative PCR as templates.
Total RNA from HDF and hiPS cells (clone 201B) was labeled with Cy3. Samples were hybridized with
Whole Human Genome Microarray 4 x 44K (G4112F, Agilent), with the one color protocol. Arrays were
scanned with a G2565BA Microarray Scanner System (Agilent). Data analyzed by using GeneSpring
GX7.3.1 software (Agilent). The microarray data of hES H9 cells (Tesar et al., 2007) was retrieved from
GEO DataSets (GSM194390,
http://www.ncbi.nlm.nih.gov/sites/entrez?db=gds&cmd=search&term=GSE7902). Genes with "present"
flag value in all three samples were used for analyses (32266 genes). We have deposited the
microarray data of HDF and hiPS cells to GEO DataSets with the accession number GSE9561.
We thank Dr. Deepak Srivastava for critical reading of the manuscript, Gary Howard and
Stephen Ordway for editorial review, Drs. Masato Nakagawa, Keisuke Okita and Takashi
Aoi and other members of our laboratory for scientific comment and valuable discussion,
Dr. Peter. W. Andrews for SSEA-3, TRA-1-60 and TRA-1-81 antibodies, and Dr. Toshio
Kitamura for retroviral system. We are also grateful to Aki Okada for technical support and
Rie Kato and Ryoko Iyama for administrative supports. This study was supported in part by
a grant from the Program for Promotion of Fundamental Studies in Health Sciences of
NIBIO, a grant from the Leading Project of MEXT, a grant from Uehara Memorial
Foundation, and Grants-in-Aid for Scientific Research of JSPS and MEXT.
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Figure 1 Induction of iPS Cells from Adult HDF
(A) Time schedule of iPS cell generation.
(B) Morphology of HDF.
(C) Typical image of non-ES cell–like colony.
(D) Typical image of hES cell-like colony.
(E) Morphology of established iPS cell line at passage number 6 (clone 201B7).
(F) Image of iPS cells with high magnification.
(G) Spontaneously differentiated cells in the center part of human iPS cell colonies. (H–N)
Immunocytochemistry for SSEA-1 (H), SSEA-3 (I), SSEA-4 (J), TRA-1-60 (K), TRA-1-81
(L), TRA-2-49/6E (M), and Nanog (N). Nuclei were stained with Hoechst 33342 (blue).
Bars = 200 μm (B–E, G), 20 μm (F), and 100 μm (H–N).
Figure 2 Expression of hES Cell Marker Genes in Human iPS Cells
(A) RT-PCR analysis of ES cell marker genes. Primers used for Oct3/4, Sox2, Klf4, and
c-Myc specifically detect the transcripts from the endogenous genes, but not from the
(B) Western blot analysis of ES cell marker genes.
(C) Quantitative PCR for expression of retroviral transgenes in human iPS cells, HDF, and
HDF six days after the transduction with the four retroviruses (HDF/4f-6d). Shown are the
averages and standard deviations of three independent experiments. The value of
HDF/4f-6d was set to 1 in each experiment.
(D) The global gene expression patterns were compared between human iPS cells (clone
201B7) and HDF, and between human iPS cells and hES cells (H9) with oligonucleotide
DNA microarrays. Arrows indicate the expression levels of Nanog, endogenous Oct3/4 (the
probe derived from the 3' untranslated region, which dose not detect the retroviral
transcripts), and endogenous Sox2. The red lines indicate the diagonal and five-fold
changes between the two samples.
Figure 3 Analyses promoter regions of development-associated genes in human iPS
(A) Bisulfite genomic sequencing of the promoter regions of OCT3/4, REX1 and NANOG.
Open and closed circles indicate unmethylated and methylated CpGs.
(B) Luciferase assays. The luciferase reporter construct driven by indicated promoters were
introduced into human iPS cells or HDF by lipofection. The graphs show the average of the
results from four assays. Bars indicate standard deviation.
(C) Chromatin immunoprecipitation of histone H3 lysine 4 and lysine 27 methylation.
Figure 4 High Levels of Telomerase Activity and Exponential Proliferation of Human
(A) Detection of telomerase activities by the TRAP method. Heat-inactivated (+) samples
were used as negative controls. IC = internal control.
(B) Growth curve of iPS cells. Shown are averages and standard deviations in
Figure 5 Embryoid Body–Mediated Differentiation of Human iPS Cells
(A) Floating culture of iPS cells at day 8.
(B–E) Images of differentiated cells at day 16 (B), neuron-like cells (C), epithelial cells (D),
and cobblestone-like cells (E).
(F–K) Immunocytochemistry of alpha-fetoprotein (F), vimentin (G), -smooth muscle actin
(H), desmin (I), βIII-tubulin (J), and GFAP (K).
Bars = 200 μm (A, B) and 100 μm (C–K). Nuclei were stained with Hoechst 33342 (blue).
(L) RT-PCR analyses of various differentiation markers for the three germ layers.
Figure 6 Directed Differentiations of Human iPS Cells
(A) Phase contrast image of differentiated iPS cells after 18 days cultivation on PA6.
(B) Immunocytochemistry of the cells shown in A with βIII-tubulin (red) and tyrosine
hydroxylase (green) antibodies. Nuclei were stained with Hoechst 33342 (blue).
(C) RT-PCR analyses of dopaminergic neuron markers.
(D) Phase contrast image of iPS cells differentiated into cardiomyocytes.
(E) RT-PCR analyses of cardiomyocyte markers.
Bars = 200 μm (A, D) and 100 μm (B).
Figure 7 Teratoma Derived from Human iPS Cells
Hematoxylin and eosin staining of teratoma derived from iPS cells (clone 201B7). Cells
were transplanted subcutaneously into four parts of a SCID mouse. A tumor developed
from one injection site.
ES medium + bFGF
Click here to download [F] Figure: Takahashi1.pdf
[F] Figure 1
1 2 3 6 7NTERA-2 HDF
2 6 713
1 2 3 6 7ES
1 2 3 6 7NTERA-2HDF
1 2 3 6 7 NTERA-2
1 2 3 6 7 NTERA-2
[F] Figure 2
Click here to download [F] Figure: Takahashi2.pdf
Click here to download [F] Figure: Takahashi3.pdf
Percentage of Input
Luciferase activity (FL/RL)
Luciferase activity (FL/RL)
Luciferase activity (FL/RL)
[F] Figure 3
- + - + - + - + - +
Cell number (log10)
[F] Figure 4
Click here to download [F] Figure: Figure 4.pdf
Click here to download [F] Figure: Takahashi5.pdf
201B2 201B6 201B7
[F] Figure 5
201B2 201B6 201B7
U D U D U D
201B2 201B6 201B7
[F] Figure 6
Click here to download [F] Figure: Figure 6.pdf
Gut-like epithelium MuscleEpidermis
Adipose tissue Neural tissue
[F] Figure 7
Click here to download [F] Figure: Takahashi7.pdf
List of supplemental online materialsList of supplemental online materials
File name: S-InfoFile name: S-Info
S-Figure 1 Improved transduction efficiency of retroviruses in HDF
S-Figure 2 Feeder Dependency of human iPS cells
S-Figure 3 Genetic analyses of human iPS cells
S-Figure 4 Human iPS cells derived from fibroblast-like synoviocytes and BJ fibroblasts
S-Figure 5 Expression of ES cell marker genes in iPS cells derived from HFLS and BJ
S-Figure 6 Embryoid body-mediated differentiation of iPS cells derived from HFLS and
S-Table 1 Summary of the iPS cell induction experiments
S-Table 2 Characterization of established clones
S-Table 3 STR analyses of HDF-derived iPS cells
S-Table 4 STR analyses of HFLS-derived iPS cells
S-Table 5 STR analyses of BJ-derived iPS cells
S-Table 6 Primer sequences
File name: S-Table 7File name: S-Table 7
S-Table 7 Genes showing more than five-fold expression in human iPS cells than in hES
File name: S-Table 8 File name: S-Table 8
S-Table 8 Genes showing more than five-fold expression in hES cells than in human iPS
File name: S-movieFile name: S-movie
Beating cardiomyoctes derived from human iPS cells
[G] List of SOM
Click here to download [G] Supplemental Text and Figures: List of SOM_Yamanaka.doc
[G] Supplemental Text and Figures
Click here to download [G] Supplemental Text and Figures: S-info_Yamanaka.doc
S-Figure 5 Download full-text