FORUM REVIEW ARTICLE
Induced Pluripotent Stem Cells:
Emerging Techniques for Nuclear Reprogramming
Ji Woong Han and Young-sup Yoon
Introduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, can successfully reprogram somatic
cells into embryonic stem (ES)-like cells. These cells, which are referred to as induced pluripotent stem (iPS) cells,
closely resemble embryonic stem cells in genomic, cell biologic, and phenotypic characteristics, and the creation
of these special cells was a major triumph in cell biology. In contrast to pluripotent stem cells generated by
somatic cell nuclear-transfer (SCNT) or ES cells derived from the inner cell mass (ICM) of the blastocyst, direct
reprogramming provides a convenient and reliable means of generating pluripotent stem cells. iPS cells have
already shown incredible potential for research and for therapeutic applications in regenerative medicine within
just a few years of their discovery. In this review, current techniques of generating iPS cells and mechanisms of
nuclear reprogramming are reviewed, and the potential for therapeutic applications is discussed. Antioxid. Redox
Signal. 15, 1799–1820.
determine what factors contribute to their plasticity and
differentiation ability. The first somatic cell nuclear-transfer
(SCNT) experiments were performed more than 50 years ago
from a differentiated blastula cell into an enucleated frog
oocyte could give rise to a tadpole. Later, Gurdon (45, 46)
showed that nuclei from even more differentiated frog intes-
tinal cells could give rise to adult animals, although the effi-
ciency was very low. These experiments showed that when
placed in the appropriate environment, the nuclei of differ-
entiated adult cells retain nuclear plasticity similarly to those
of the early embryo. In 1996, Wilmut and colleagues (18, 195)
reported the first cloned animal, Dolly the sheep, by nuclear
transfer from adult cells into denucleated eggs. These
achievements supported earlier findings that the epigenetic
state of nuclei of differentiated cells is changeable, and that
nuclei retain the ability to be reprogrammed by factors in
oocytes or embryonic stem (ES) cells.
Pluripotency is the ability of the cell to differentiate to all
cell types of an adult organism (126). Pluripotency occurs
naturally only in early embryos and may be maintained
in vitro in cultured ES cells harvested from the ICM of blas-
tocysts. Isolated ES cells can maintain their population by
proliferating and self-renewing indefinitely, and have the
potential to differentiate into every lineage type in the body
(38, 108). Self-renewal allows ES cells in culture to undergo
cientists have long experimented with mature cells to
numerous cell cycles, including cell division, without losing
pluripotency under specific conditions (38, 108). Mouse ES
unknown but essential factors. The culture medium must also
contain leukemia inhibitory factor (LIF) for mouse ES cells, or
fibroblast growth factors (FGFs) for human ES cells, to pre-
vent differentiation (176). Without feeders or cytokines, ES
cells undergo spontaneous differentiation and lose their
pluripotency. Nuclear reprogramming, the process used to
make induced pluripotent stem (iPS) cells, is the reverse of
differentiation, in which differentiated cells revert to plurip-
otent cells (35, 63).
cells therapeutically for treating patients. Both human ES cell
and somatic cell nuclear transfer (SCNT) have technical and
ethical problems that make therapeutic use in humans diffi-
cult. iPS cell technology circumvents these problems and is
regarded as the best method for generating patient-specific
pluripotent stem cells for use in regenerative medicine.
Generation of Induced Pluripotent
Stem Cells from Somatic Cells
Generation of pluripotent stem cells from adult cells is an
artificial manipulation that may not produce cells identical to
naturally occurring pluripotent stem cells. However, some
aspects of iPS cell generation may parallel the innate genetic
processes that occur during embryonic development, in-
cluding the reprogramming of the gamete pronuclei at
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
ANTIOXIDANTS & REDOX SIGNALING
Volume 15, Number 7, 2011
ª Mary Ann Liebert, Inc.
fertilization under the influence of factors in the oocyte.
Although tremendous effort has been put into generating
immune-compatible patient-specific stem cells, promising
methods were not successful until iPS cell technology with
defined transcription factors was developed (172) (Table 1).
Ectopic overexpression of transcription factors
Based on the success and limitations of previous nuclear-
transfer and cell-culture experiments, scientists began to
experiment with more direct manipulation of cells’ genetic
information to create developmental plasticity in mature, dif-
ferentiated cells. The first successful generation of iPS cellsfrom
somatic cells was accomplished by ectopic overexpression of
pluripotency-related transcription factors (172). Takahashi and
Yamanaka (172) introduced a mini-library of 24 candidate re-
which were known to be expressed in ES cells. The genes were
introduced into mouse embryonic fibroblasts (MEFs) that car-
ried a fusion of the b-galactosidase and neomycin-resistance
genes expressed from the Fbx15 locus (177). When MEFs were
medium in the presence of G418, drug-resistant colonies
emerged that had ES cell–like proliferation, gene expression,
and morphology. To narrow the factors that are essential for
reprogramming, all combinations of the 24 factors were at-
tempted until four factors, Oct3/4, Sox2, Klf4, and c-Myc, were
identified. The resultant cells, which showed pluripotent fea-
tures indistinguishable from those of ES cells, were referred to
as induced pluripotent stem (iPS) cells (172) (Fig. 1). These four
essential factors are often referred to as Yamanaka factors in
recognition of the inventor of this method, Shinya Yamanaka.
After this discovery, several groups have improved on the
original reprogramming method. One group combined Oct3/
4 and Sox2 with Lin28 and Nanog to derive human iPS cells
(211). Remarkably, the same four factors identified in the
murine system were able to confer pluripotency in primate
cells, indicating that the fundamental transcriptional network
governing pluripotency is conserved across species. Several
groups have shown that the c-Myc gene is dispensable for
reprogramming (122, 138), although efficiency was then quite
low. Recently, Yamanaka and colleagues (123) showed that c-
Myc can be replaced with L-Myc, another Myc family mem-
ber, for generating human iPS cells, resulting in even higher
efficiency. As c-Myc reactivation can trigger the tumorige-
nicity of iPS derivatives, this study is important.
Although most iPS cells were generated from skin fibro-
blasts with these reprogramming transcription factors (171,
172, 211), expression of the same reprogramming factors also
appears to initiate a sequence of stochastic events that even-
tually lead to generating iPS cells in a variety of other differ-
entiated cells, such as neural cells (72, 73, 154, 156),
keratinocytes (19), melanocytes (181), adipose-derived cells
(164, 174), amniotic cells (86, 121, 216), pancreatic cells (159),
and blood cells (50, 96, 150, 161) (Fig. 1). The forcedexpression
of the four genes is required only temporarily, at the initiation
stage of reprogramming, and can then be mostly silenced
Methods of delivering transcription factors into cells
Integrating viral vectors.
vectors, including retrovirus (132, 138, 172) and lentivirus (14,
Severaltypes ofintegrating viral
159, 190), have been used in iPS cell generation. Retroviral
vectors were the first type of vectors used to create iPS cells,
and the site of viral integration has been closely studied. Ta-
kahashi and Yamanaka (172) noted approximately 20 retro-
In a recent report comparing reprogramming of MEFs with
that of murine hepatocytes or gastric epithelial cells, Aoi and
colleagues (5) examined the number of RISs for each of the
four retroviruses by using Southern blot.They detected one to
nine RISs in MEF-derived iPS clones and one to four RISs in
gastric epithelium or hepatocyte-derived clones, suggesting
that tissues of epithelial origin may be more readily repro-
grammed. The integration sites were random and did not
show common viral integration sites. Several RISs found by
Aoi had previously been identified by retroviral-mediated
tumor induction in mice, so the safely of iPS cells is still
questionable (2, 52). Bioinformatics analysis revealed no en-
richment of any specific gene function, gene network, or ca-
nonic pathway by retroviral insertions (184). Retroviruses
have a propensity to integrate near transcription start sites
and may be more likely to cause malignant transformations.
Lentiviral vectors have a hypothetical safety advantage
over retroviral vectors, because they lack the propensity to
integrate near transcription start sites (201). No insertion-site
analysis has been conducted, and thus the biologic relevance
of vector differences remains theoretic. An additional ad-
vantage of lentivirus is its ability to transcribe large genetic
packages, and two recent publications detailed the use of
polycistronic lentiviral vectors that delivered the four repro-
gramming factors in a single construct, instead of the four
separate vectors, each carrying one gene, used previously (19,
157). Both articles demonstrate the derivation of iPS clones
from a single vector integration, which may minimize muta-
Nonintegrating viral vectors.
germline-competent iPS cells, *20% of chimeric mice had
tumors most likely caused by reactivation of the integrated
c-Myc proviral transgene in the host genome (132). Another
study showed cancer-related mortality in 18 (50%) of 36 of iPS
chimeric mice (191). Insertional mutagenesis due to the inte-
gration of viral vectors into critical sites of the host genome,
leading to malignant transformation, has been observed in
preclinical and clinical gene therapy trials (48, 58, 91). Because
of these limitations and safety concerns, alternative methods
of iPS cell generation have been sought and have focused on
eliminating integration of retroviral and lentiviral vectors
from the reprogramming procedure.
Accordingly, the potential for using nonintegrating vectors
was explored. As it is known that only transient expression of
the four original factors is required. Stadtfeld and colleagues
(160) used a transiently active and nonintegrating adenoviral
vector and succeeded in generating iPS cell lines. These
viruses could contribute to the formation of teratomas and
chimeric mice, but were unable to pass through the germline
(160). By contrast, Okita and colleagues (133) were unable to
obtain murine hepatocyte iPS clones when the four repro-
gramming factors were introduced by adenovirus alone and
required additional transfections of Oct3/4 and Klf4 or Oct3/4
and Sox2 by retrovirus. For unknown reasons, some cell types
may be amenable to the safer adenoviral vector transduction,
whereas other cell types cannot be made into iPS cells by using
In the first report on
1800HAN AND YOON
Table 1. The Methods of iPS Cell Generation
SpeciesMethodsTFs Cell types Other factorsRef.
Amniotic, yolk-sac cells
Adipose-derived stem cells
Fibroblasts, p53-/-, Terc-/-
miR-291-3p, 294, 295
p53, p21, Ink4a/Arf
Pancreatic b cells
vectors (tet), single
OSKMFibroblasts, hepatocytes (160)
Liver epithelial cells
lentiviral vectors (tet)
hTERT, Large T, ROCKi
INDUCED PLURIPOTENT STEM CELLS1801
a fundamental but unanswered question in stem cell biology.
generating iPS cells with adenovirus suggests that this safer
vector may be useful, the widespread use of any viral-vector
technology in human application is likelyimpossible. Because
of the great potential of iPS cells, investigators have turned to
nonviral vector systems for the generation of iPS cells. Ya-
manaka and colleagues (133) used a polycistronic expression
plasmid containing the Oct3/4, Sox2, and Klf4 cDNAs linked
by the foot-and-mouth disease virus 2A self-cleaving peptide.
When this construct, which lacks viral genetic material, was
repeatedly transfected into MEFs, together with a separate c-
Myc cDNA expression vector over a 1 week time period (on
days 1, 3, 5, and 7), one to 29 Oct4-positive iPS colonies
emerged from 1·106cells in seven of 10 independent exper-
iments, whereas from the same number of retrovirus-infected
cells, 100 Oct4-positive iPS colonies were routinely obtained.
Although the partial success in
In six of 10 experiments, no evidence of plasmid integration
into the host genome was detected with PCR or Southern blot
analysis (133). Although the efficiency of iPS generation by
plasmid transfection was greatly decreased, and the onco-
genic c-Myc viral vector transduction was needed, this report
provided proof-of-concept for the generation of iPS cells
without transgene integration of viral vector.
Several recent studies have reported the use of a transient
viral-vector approach to iPS generation that would increase ef-
ficiency and safety (66, 197, 212). This method begins with the
incorporation of all four Yamanaka genes into a single piggyBac
allowing synthesis of the four factors as a single transcript fol-
lowed by posttranslational cleavage of the proteins at the ap-
propriate locations. The most important and unique feature of
this approach is that the reprogramming genes are removed
from the genome by transient transfection of PB transposase
(212). Thus, permanent alteration of the genome is avoided and
Table 1. (Continued)
SpeciesMethodsTFs Cell types Other factors Ref.
Fibroblasts from patients
p53 shRNA, p53DD(56)
Fibroblasts, oral mucosa
Amniotic fluid cells
Amniotic/yolk sac cells
Cord blood cell, CD34+
Cord blood cells, CD34+
Blood cell, PB-and BM–MNC
Cord blood cells, CD45-
Fibroblasts from patients
Mesenchymal stromal cells
Cord blood cells, CD133+
p53 shRNA, p53DD
Dermal papilla cells
Fibroblasts from patients
Cord blood cells
Blood cell, T cell/PB–MNC
Blood cell, PB–MNC
Lentiviral vectors Large T
vectors (tet), single
OSKM/OSKFibroblasts from patients (156)
Blood cell, T cell
O, Oct4; S, Sox2; K, Klf4; M, c-Myc; N, Nanog; L, Lin28;LM, L-Myc.
1802HAN AND YOON
relatively robust reprogramming efficiency. By using a similar
idea, the Cre–loxP recombinase system was used to remove the
vector-integrated transgene once reprogramming is achieved
(66). However, this system is less precise and may leave some
residual elements outside the loxP sites, including the transpo-
transient expression of the reprogramming factors is sufficient
for reprogramming of somatic cells to acquire pluripotency.
They also confirmed that it is possible to remove integrated
vector material when it is no longer needed and thus to mini-
mize the risk of late cancer development.
Recombinant proteins of transcription factors.
ternate approach, the protein products of the reprogramming
at all (69, 219). Two independent groups made proteins in
which reprogramming factors were fused to polyarginine, a
short basic peptide, known as a cell-penetrating peptide
(CPP), which can overcome the cell-membrane barrier and
As an al-
colleagues (219) used purified recombinant proteins contain-
ing 11 arginine residues (11R) at the C-terminus of each re-
programming factor to reprogram OG2/Oct4-GFP reporter
MEF cells with four cycles of protein treatments plus the
histone deacetylase (HDAC) inhibitor, valproic acid (VPA).
fetal fibroblasts by treatment of cell extracts from HEK293
cells, which expressed each reprogramming factor fused to
eight arginine residues (8R), albeit at a very low efficiency
(*0.001%) and with prolonged time (*8 weeks). A recent
study by Cho and colleagues (26) also is encouraging, as they
reported that a single transfer of ES cell–extracted proteins to
mouse fibroblasts with cell permeabilization can successfully
reprogram mouse fibroblasts in relatively short time periods
(20*25 days after induction), albeit at a still lower efficiency
(0.001%). Thus, although in its present form, this protein-
based reprogramming technique may fall short of generat-
ing patient-specific iPS cells, it is the only promising and
realistic technology at the moment to generate virus-free and
transgene-free human patient–specific iPS cells.
embryonic fibroblasts in mouse and skin fibroblasts in humans. Fibroblasts have been most widely used for reprogramming.
Thereafter, other cells, including neural stem cells, keratinocytes, melanocytes, adipose tissue–derived cells, and various
blood mononuclear cells, have been successfully used for reprogramming to achieve higher efficiency or to show easier and
more convenient accessibility. Advances have occurred in the delivery vectors for reprogramming factors, Oct4, Sox2, Klf4,
c-Myc, Nanog, and Lin28. Initial studies used DNA-integrating viral vectors such as retrovirus and lentivirus, but later studies
demonstrated the usefulness of nonintegrating viral vectors, adenovirus, and nonviral systems with plasmids for repro-
gramming. More recently, delivery methods using recombinant proteins or ES-cell extracted proteins have been shown to
reprogram somatic cells into iPS cells, suggesting safer and potentially clinically applicable systems for deriving iPS cells. (To
see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
Various cell sources and delivery methods for generating iPS cells. iPS cells were originally derived from
INDUCED PLURIPOTENT STEM CELLS1803
Nongenetic approaches for reprogramming
the effects of small-molecule chemicals involved in chromatin
modification on reprogramming by using Oct4-GFP reporter
mouse cells. Treatment of the four factor-infected MEFs with
the DNA methyltransferase inhibitor 5¢-azacytidine increased
the reprogramming efficiency, and three known histone
deacetylase (HDAC) inhibitors—suberoylanilide hydroxamic
acid (SAHA), trichostatin A (TSA), and valproic acid (VPA)—
also greatly increased the efficiency of reprogramming, with
VPA being the most effective compound, showing a >100-
fold increase (59). This demonstration of enhanced repro-
gramming efficiency by HDAC inhibitors suggests that
chromatin modification is a key step in defining the pluripo-
tent state of a cell.
BIX01294 and BayK8644, which, in combination with two
factors (Oct4 and Klf4), enhanced the reprogramming effi-
ciency of mouse neural progenitors (153) and mouse embry-
onic fibroblasts (152). BIX01294 is an inhibitor of the G9a
histone methyltransferases, which methylate histone H3 at
the position of lysine 9 (H3K9) (24). G9a histone methyl-
transferase is reported to silence Oct4 expression during early
embryogenesis by subsequent de novo DNA methylation at
the promoter region, thereby preventing reprogramming (30,
36, 39, 170).
Several other small molecules have been found that indi-
rectly influence the epigenetic state of a cell (Fig. 2). BayK8644
is an L-channel calcium agonist (153) that exerts its effect
through upstream signaling pathways rather than direct
epigenetic remodeling. Other small molecules involved in
signaling pathways also increase the efficiency of repro-
gramming, such as rho-associate kinase (ROCK) inhibitor,
Y-27632, which augments human iPS cell induction by
enhancing cell survival (77, 138). Inhibitor of Wnt signaling
(89, 107, 155), MEK (89, 155), FGF (66), and TGF-b receptor
(61, 89) also had effects on the generation and maintenance of
ground-level pluripotency of iPS cells.
potential of microRNA (miRNA) for the production of iPS
cells. Pioneering studies revealed the existence of subsets of
miRNAs, which are specific for ES cells, referred to as ES cell-
specific cell cycle–regulating miRNAs (ESCC miRNAs) (188).
Transient transfection of ESCC miRNAs (miR-291-3p, miR-
294, and miR-295), in combination with retroviral infection
expressing Oct4, Sox2, and Klf4, showed enhanced efficiency
of iPS cell generation from mouse embryonic fibroblasts (65).
Efforts have been made to investigate the
deacetylase (HDAC) inhibitors, and histone methyltransferase inhibitor were shown to increase the reprogramming effi-
ciency by epigenetic remodeling of pluripotency-related genes. L-channel calcium agonist, rho-associated kinase (ROCK)
inhibitor, Wnt signaling inhibitor, and inhibitors of MEK, FGF, and TGF-b receptor can enhance reprogramming efficiency.
(To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
Small-molecule chemicals for enhancing reprogramming efficiency. DNA methyltransferase inhibitor, histone
1804 HAN AND YOON
Although it remains unclear whether these miRNAs have
overlapping targets during reprogramming, studying their
regulation during the iPS cell-generation process will enhance
understanding of the biology of miRNAs associated with
pluripotency. It would also be interesting to test whether the
inhibition of miRNAs such as let-7 and mir-125 (196), ex-
pressed in only differentiated cells but not in stem cells, fa-
cilitates reprogramming. Recently, Melton and colleagues
(114) showed that introduction of let-7 inhibitors with retro-
viral vectors expressing Oct4, Sox2, and Klf4 (minus c-Myc)
increased the efficiency of reprogramming by 4.3-fold. To-
gether, these findings indicated an important role of opposing
miRNA families, ES-specific, and differentiated cell-specific
miRNAs, on reprogramming capacity and their potential as
Altering cell-cycle signal pathways.
proteins canalso changetheefficiencyofiPScell generation in
ways that are just beginning to be understood. Inhibition of
the tumor-suppressor protein p53–related signal pathway
facilitates the generation of iPS cells, suggesting that it re-
presses the dedifferentiation (56, 68, 182). iPS cell technology
can be used to study how p53 modulates the stability of the
differentiated state. Inhibition of p53 directly by Mdm2 and
indirectly by downregulation of Arf (87, 182) enhances the
progression of normal fibroblasts into iPS cells through its
direct target, p21, which promotes cellular senescence (56, 68).
Deficiency of p53 improves the efficiency and kinetics of iPS
cell generation with only two factors, Oct4 and Sox2 (68).
Other tumor suppressors, such as Rb or Pten, are also candi-
date repressors of dedifferentiation that can be investigated
by using iPS cell technology (218).
Advances in the source cells
The choice of appropriate cell types for nuclear repro-
gramming is not only important but also critical for future
clinical therapy with autologous iPS cells; therefore, an easy
sampling method with minimal risk and yielding a sufficient
quantity of cells for reprogramming must be developed.
Originally, skin fibroblasts were chosen as source cells for
reprogramming because these cells are relatively easy to col-
lect and expand in in vitro culture. However, procuring these
cells from patients necessitates a skin biopsy, requiring sur-
gical procedures, such as anesthesia, incision, and suturing,
which are inconvenient, painful, and have potential compli-
cations, including infection. Notwithstanding this, iPS cells
have been established from skin fibroblasts derived from
patients with a variety of diseases, including adenosine de-
aminase (ADA) deficiency, Gaucher disease type III (GD),
Duchenne muscular dystrophy (DMD), Becker muscular
dystrophy (BMD), Down syndrome (DS), Parkinson disease
(PD), juvenile diabetes mellitus (JDM), Huntington disease
(HD), Lesch-Nyhan syndrome (LNS), amyotrophic lateral
Fanconi anemia (FA), and LEOPARD syndrome (21, 29, 33,
137, 142, 156, 189). Other cell types have advantages over skin
fibroblasts. Because they have a higher expression level of
endogenous pluripotency-related genes Sox2 and c-Myc,
neural stem cells (NSCs) can be reprogrammed by a smaller
number of reprogramming factors, two factors (Oct4 and
Klf4), or even just one factor (Oct4) (54, 71, 72, 153). Kerati-
nocytes derived from human foreskin and plucked hairs,
which express high levels of c-Myc, showed *100-fold im-
provement in reprogramming efficiency over fibroblasts (1).
Human melanocytes from skin biopsies express high levels of
endogenous Sox2 and can be reprogrammed with three fac-
tors (Oct4, Klf4, and c-Myc) with higher efficiency (0.05%) and
more quickly (*10 days) (181). Adipose-derived stem cells,
which can be obtained from routinely performed lipoaspira-
tion in outpatient clinics, were reprogrammed in a feeder-free
system by four factors (Oct4, Klf4, Sox2, and c-Myc) with *20-
fold higher efficiency and about twofold faster (167). CD133+
cells from frozen banked cord blood can be reprogrammed
with only two factors, Oct4 and Sox2 (42), or Thomson factors
(Oct4, Sox2, Nanog, and Lin28) (47). In addition, granulocyte
colony-stimulating factor (G-CSF) mobilized CD34+cells
from peripheral blood were used as a cell source to generate
iPS cells with four factors (95, 206). However, the CD34 frac-
tion represents a very rare population in peripheral blood,
and therefore, isolation of a sufficient amount of CD34+cells
requires the use of expensive G-CSF and a large-volume
apheresis. Other groups, therefore, have recently developed
new methods by using a small amount of whole peripheral
blood as a better source of cells for human patient-specific iPS
cell generation (96, 150, 161) (Fig. 1).
Each type of source cell has its own advantages and limi-
tations for the generation of iPS cells. In addition, recent
studies have shown the significant effects of genetic (41) and
epigenetic (73) memory on characteristics and differentiating
potency among iPS cell lines, depending on the types of
source cells. If these results are further verified by more
studies, the choice of the optimal cell source for patient-
specific iPS cell generation may be carefully considered and
selected for each specific purpose.
Characterization of iPS Cells
iPS cells express ES cell–specific genes that maintain the
developmental potential to differentiate into all three primary
germ layers: ectoderm, endoderm, and mesoderm. Several
functional tests, including in vitro differentiation, DNA
methylation analysis, in vivo teratoma formation, chimera
formation, germline transmission, and tetraploid comple-
mentation, have been used to define pluripotency of iPS cells.
Generally, ES cells and iPS cells seem to have almost identical
properties in these assays (67, 217).
Maintaining genomic integrity is of crucial importance
during the creation of iPS cells, as alterations can cause neo-
plastic disease and limit therapeutic application. Several
groups have investigated the karyotypes of mouse (192) and
alteration is present. One study showed that continuous
passaging of human iPS cells resulted in chromosomal ab-
normalities, starting as early as passage 13 (1). This finding
warns that more studies should investigate the exact fre-
quency of culture-induced genetic abnormalities in human
iPS cells over the long run.
The use of retroviral and lentiviral vectors to express the
reprogramming transcription factors has the inherent risk of
insertional mutagenesis. However, Aoi and colleagues (5)
found no common insertion sites in hepatocyte- and stomach
INDUCED PLURIPOTENT STEM CELLS1805
cell–derived iPS cells (5). In addition, recent adenoviral and
plasmid-based methods have a much lower risk of insertional
mutagenesis, because theoretically, the genomic DNA is not
perturbed by the virus (133, 160). However, even without
viral integration, genetic changes might occur as part of the
reprogramming process. Another component reflecting DNA
integrity, telomere length, is not altered in mouse (160) and
human hTERT (138, 171)-derived iPS cells (171). Studies of iPS
cells have suggested that DNA integrity is maintained
throughout the generation process; however, only long-term
studies will show if these cells are truly free of malignant
potential in vivo. In any case, this risk will have to be weighed
against the therapeutic potential.
Comparative global gene-expression analyses of the ES cell
and iPS cell transcriptomes by using microarrays have been
performed for human and mouse lines (103, 172, 192). Mik-
kelsen and colleagues (116) reported that whole-genome ex-
pression profiles of iPS cells and ES cells of the same species
are no more different than are those of individual ES cell lines.
to ES cells. Takahashi and colleagues (171) compared the
global gene-expression profile of human iPS and human ES
cells for 32,266 transcripts. Notably, 1,267 (*4%) of the genes
were detected with more than fivefold difference in up- or
downregulation between iPS cells and human ES cells. Sold-
ner and colleagues (156) compared the transcriptional profiles
of human iPS cell lines, in which the Cre-recombinase excis-
human iPS cells), with those of human iPS cells before trans-
gene excision. The transcriptomes of factor-free human iPS
cells more closely resembled those of human ES cells than the
parental human iPS cells with integrated viral sequences. This
could be caused by the loss of any downstream gene activa-
tion by residual expression of the exogenous transcription
after the initial reprogramming event. However, it remains
difficult to compare these differences because most groups
used genetically unrelated cell lines. More experiments are
needed to clarify these discrepancies according to the meth-
of parental cells, ages, and sex.
A well-characterized gene-expression pattern occurs after
ectopic expression of the four factors in mouse embryonic
fibroblasts, including an initial downregulation of cell-type–
specific transcription factors (116, 159) and upregulation of
genes involved in proliferation, DNA replication, and cell-
cycle progression (116). During the reprogramming process,
many self-renewal–related genes are reactivated, including
fibroblast growth factor 4 (Fgf4) as well as polycomb genes
(116, 172). However, a large fraction of pluripotency-related
genes are upregulated only during the late stages of repro-
key pluripotency-related genes, such as Oct4, Sox2, and Rex1,
was approximately twofold lower in the iPS cells compared
with two human ES cell lines, HSF1 and H9 (98). Pluripotent
cells are highly sensitive to the levels of these transcription
factors (TFs) (127), and a notable amount of normal tran-
scriptional heterogeneity is found in human ES-cell cultures
(135). Therefore, the observed variation could reflect differ-
ences in culture conditions rather than incomplete repro-
gramming. More work on human ES cells is thus required the
better to understand the extent of normal transcriptional
variation within human and also mouse ES cells and fully to
understand how iPS cells compare.
As the substrate of transcription, chromatin is subjected to
various forms of epigenetic regulation, including chromatin
remodeling, histone modifications, histone variants, and
lysine 27 of histone 3 (H3K9 and H3K27) correlates with in-
active regions of chromatin, whereas H3K4 trimethylation,
and acetylation of H3 and H4 are associated with active
transcription (64), and DNA methylation generally represses
gene expression (145).
By regulating chromatin structure, epigenetic modifica-
tions play an essential role in controlling access to genes and
regulatory elements in the genome (14). The differences in
epigenetic status between a somatic cell and a pluripotent
stem cell are huge, and dedifferentiation requires global epi-
genetic reprogramming. For instance, pluripotent stem cells
contain bivalent domains that are characteristic chromatin
signatures (9, 10). These are regions enriched for repressive
histone H3 lysine 27 trimethylation (H3K27me3) and simul-
taneously for histone H3 lysine 4 trimethylation (H3K4me3)
as an activating signal (117). It was assumed initially that
bivalent domains might be ES-cell specific because they were
first identified by using chromatin-immunoprecipitation
(ChIP) followed by hybridization to microarrays (ChIP-Chip)
that featured key developmental regulators. All of these re-
solved either to a univalent (H3K4me3 only or H3K27me3
only) state or lost both marks in differentiated cells (9). With
ChIP followed by high-throughput sequencing (ChIP-seq)
technology, Mikkelsen and colleagues (112) showed that bi-
valent domains are more generally indicative of genes that
remain in a poised state. Consequently, pluripotent cells were
found to contain large numbers of bivalent domains (*2,500)
compared with multipotent neural progenitor cells (NPCs)
(*200) that still retain multilineage potential but are more
restricted than ES cells (112).
Several studies of the murine iPS-cell have identified a
small number of representative loci that have consistent
chromatin and DNA methylation patterns (103, 172, 192).
Maherali and colleagues (103) used ChIP-Chip to investigate
the presence of H3K4me3 and H3K27me3 in the promoter
highly similar to ES cells in epigenetic state (103). The
H3K4me3 pattern was similar across all samples, indicating
that reprogramming was largely associated with changes in
H3K27me3 rather than with H3K4me3 (103). Mikkelsen and
colleagues (192) used a more-comprehensive ChIP-Seq tech-
nique to determine genome-wide chromatin maps in several
iPS lines, which are derived with different methods: drug
selection by using an Oct4–neomycin-resistance gene (192),
drug selection by using a Nanog–neomycin-resistance gene
(192), and by morphologic appearance (113). Overall global
levels of repressive H3K27me3 and the characteristic bivalent
chromatin structure are retained in the various iPS cell lines.
The restoration ofrepressive chromatin marksappearscrucial
stably to silence lineage-specific genes that are active in
1806HAN AND YOON
Failure to establish the repressive marks results in incom-
pletely reprogrammed cells. Activating H3K4me3 patterns
are also crucial for complete reprogramming and have been
observed to be restored genome-wide, in particular around
the promoters of pluripotency-associated genes, such as Oct4
and Nanog, in the fully reprogrammed iPS lines (116).
A second component of the epigenetic machinery is DNA
methylation, which is a stable and heritable mark that is in-
volved in gene silencing, including genomic imprinting and
X-chromosome inactivation. DNA methylation patterns are
dynamic during early embryonic development and are es-
sential for normal postimplantation development (144).
Overall, DNA methylation levels remain stable during ES-cell
differentiation, although they are not static for any given in-
dividual gene (112). The 5’-promoter regions of many tran-
scriptional units contain clusters of the dinucleotide CpG,
which are methylated at transcriptionally silent genes and
demethylated on activation. In differentiated cells, the Oct4,
Nanog, and Sox2 promoter regions are highly methylated and
in an inactivated state, whereas in ES cells, these promoters
are unmethylated to be activated. During reprogramming,
almost complete demethylation of these promoters has been
observed (103, 116, 132, 192). Therefore, the loss of DNA
methylation at the promoters of pluripotency-related genes
appears essential for achieving complete reprogramming.
Interestingly, loss of DNA methylation at this class of genes
seems to be a rather late event in the reprogramming process
because cells that have already acquired self-renewing prop-
erties still showed high levels of DNA methylation (116).
Developmental potential: pluripotency
Research on the transcriptional and epigenetic state of iPS
cells is highly informative, and it might ultimately be possible
to characterize newly derived iPS cell lines based on their
markers, it is important to use in vivo assays to analyze the
interplay between transcriptome, epigenome, and develop-
mental potential. Recently, Jaenisch and Young (63) provided
a detailed comparison of the different strategies for assessing
developmental potential and their stringency. In vitro differ-
entiation is the least stringent assay, whereas tetraploid-em-
bryo complementation is the most stringent assay for testing
developmental potential (34, 63). These strategies could be
used to determine the pluripotency of mouse iPS cells, but
only in vitro differentiation and teratoma formation could be
developmental potential similar to that of ES cells, as con-
firmed by teratoma formation capability and by a high con-
tribution to chimera formation with germline transmission
(103, 172, 192). To show the final step of developmental po-
tential of iPS cells as equivalent to that of ES cells, three sep-
arate groups injected mouse iPS cells (2N) into tetraploid
blastocysts (4N), which are capable of producing placental
and other extraembryonic tissues but not the embryo itself,
and have created live mouse (11, 67, 217). The procedure,
called tetraploid complementation, is the most stringent test
for pluripotency. If the stem cells that are injected into the
tetraploid blastocyst differentiate into embryonic tissues that
produce a mouse, then the stem cells are considered truly
Mechanism of Reprogramming
Whereas Oct4, Sox2, Klf4, and c-Myc are known to generate
mouse and human iPS cells, another study reported that Nanog
and Lin28 (211) can replace Klf4 and c-Myc. Therefore, Oct4 and
Sox2 appear to be key transcription factors in iPS cell generation,
and the complexity of downstream events maintains plur-
ipotency and blocks differentiation (127). Several hundred
downstream genes of Oct4 and Sox2 exist with complex inter-
Klf4 and c-Myc, are known to promote cellular proliferation,
chromatin remodeling, and prevention of cell death (205). Stu-
dies from Yamanaka and Daley (138, 171) used different com-
binations of three to six genes to reprogram cells, adding SV40
c-Myc (138). Lin28 is a regulator of microRNAs previously
(143). Lin28 appears to function by inhibiting a let-7 microRNA
cellular proliferation (186). Surprisingly, many roads appear
capable of leading to the induction of pluripotency. The type of
gene appears to be of greater importance than the individual
identity of the gene used.
Transcription factor networks.
thought that the differentiation process was irreversible until
the successful cloning of Dolly by SCNT (18). This cloning
experiment demonstrated that somatic cells can be repro-
grammed back to the totipotent zygotic state by cellular fac-
tors in unfertilized eggs. The complex signaling interactions
represses itself when overexpressed, whereas Nanog, Sox2,
and FoxD3 activate its expression (127). Additional repressors
may yet be unidentified that provide counterbalance for
maintaining Oct4 expression levels in ES cells.
Based on large-scale data sets, Young and colleagues (12)
proposed that Oct4, Sox2, and Nanog collaborate to form
regulatory circuitry consisting of autoregulatory and feed-
forward loops that dictate pluripotency and self-renewal (Fig.
function collectively to maintain their own expression stably
(3). Autoregulatory loops appear to be a general feature of
master regulators of cell state (10). Functional studies have
confirmed that Oct4 and Sox2 co-occupy and activate the Oct4
and Nanog genes (80, 134), and experiments with an inducible
Sox2-null murine ES-cell line have provided compelling evi-
dence for this interconnected autoregulatory loop and its role
in the maintenance of pluripotency (109). The loop formed by
Oct4, Sox2, and Nanog also suggests how the core regulatory
circuitry of iPS cells might be triggered when Oct4, Sox2, and
other transcription factors are overexpressed in fibroblasts
(103, 132, 172, 192). When these factors are ectopically over-
expressed, they may directly activate endogenous Oct4, Sox2,
and Nanog, the products of which in turn contribute to the
maintenance of their own gene expression. Similar approaches
were attempted by other groups to identify these networks by
high-throughputtechnologies,and more elaborate mechanisms
were proposed (97, 136, 187, 220). Oct4, Sox2, and Nanog co-
occupy several hundred genes, often at apparently overlapping
genomic sites (12, 97). A large multiprotein complex containing
In mammals, it was
INDUCED PLURIPOTENT STEM CELLS 1807
ES cells (187). Other pluripotency factors may function in
complexes to control their target genes, and this phenomenon
overexpression of multiple transcription factors. Not all com-
ponents of this putative complex are required to initiate the
process of reprogramming because exogenous Nanog is not
necessary for iPS generation. It seems that exogenous Oct4 and
other factors induce expression of endogenous Nanog to levels
sufficient to accomplish full reprogramming.
The master regulatorsof
promoters of active genes encoding transcription factors,
signal-transduction components, and chromatin-modifying
enzymes that promote ES cell self-renewal (12, 97). However,
these transcriptionally active genes consist of only about half
of the targets of Oct4, Sox2, and Nanog in ES cells. These
master regulators also co-occupy the promoters of a large set
of development-specific transcription factors that are silent in
ES cells, but whose expression is associated with lineage
commitment and cellular differentiation (12, 97). Silencing of
these developmental regulators is almost certainly a key fea-
ture of pluripotency, because expression of these develop-
mental factors is associated with commitment to particular
lineages. MyoD, for example, is a transcription factor capable
of inducing a muscle gene-expression program in a variety of
cells (28). Therefore, Oct4, Sox2, and Nanog likely help main-
tain the undifferentiated state of ES cells by contributing to
repression of lineage-specification factors.
In addition to Oct4, Sox2, and Nanog, many other factors
required for pluripotency have been identified, including
Sall4, Dax1, Essrb, Tbx3, Tcl1, Rif1, Nac1, and Zfp281 (62, 97,
187). These pluripotency factors regulate each other to form a
complicated transcriptional regulatory network in ES cells
Nanog and co-occupies Nanog and Sall4 enhancer regions.
Additionally, Sall4 also regulates Oct4 expression by binding
to the Oct4 promoter (200, 214). Essrb and Rif1 are primary
targets of both Oct4 and Nanog (97).
Besides their DNA-binding activities, these pluripotency-
related proteins are extensively interconnected by protein–
protein interaction. A protein-interaction network in mouse
ES cells has been constructed by tagging Nanog and then
interactome is highly enriched for proteins that are required
for the survival or differentiation of the ICM and for early
development. Many of the genes encoding proteins in the
interaction network are targets of Nanog or Oct4 or both,
suggesting that the transcriptional network might have a
feedback mechanism through the protein-interaction net-
work. The protein-interaction network is linked to several
cofactor pathways largely involved in transcriptional repres-
sion (187). These data support a model wherein essential
factors maintain the pluripotent state by simultaneously
activating genes involved in pluripotency and repressing
genes important for development (Fig. 3).
proteins(187). This mini-
Extrinsic signal networks for maintaining pluripotency.
and iPS cells require extrinsic growth factors for maintenance
of pluripotency in culture (Fig. 4), suggesting that plur-
ipotency is an inherently unstable cell state and that ES cells
are ‘‘primed’’ for rapid differentiation. Historically, ES cells
were cultured in the presence of an underlying feeder-cell
layer of mitotically inactivated fetal fibroblast cells, which
provides an environment capable of supporting pluripotency
and blocking spontaneous differentiation. The necessary fac-
tor for self-renewal of mouse ES cells is leukemia inhibitory
the absence of the fibroblast cell feeder layer (194). LIF is not
required for pluripotency of the ICM in vivo (125) and is un-
able to maintain pluripotency in human ES cells, suggesting
that alternative mechanisms function in the maintenance of
pluripotency within these contexts. Serum is also important
for mouse ES cell maintenance, although bone morphogenic
protein 4 (BMP4) is able to replace this requirement (140, 207).
In addition, Wnt signaling has been found to act synergisti-
cally with LIF to maintain pluripotency in mouse ES cells and
appears to have a role in human ES cells (129, 146). Autocrine
loops of Activin/Nodal signaling have also been implicated
in the maintenance of mouse ES cells (129).
circuitry for nuclear repro-
gramming. The exogenous re-
programming factors, which are
eventually silenced by de novo
DNA methylation, may activate
Nanog core transcriptional regu-
latory network, which forms
a feed-forward autoregulatory
loop that positively activates the
RNA polymerase II (pol) to ini-
tiate transcription of the self-
renewal maintaining genes and
negatively to repress the differ-
entiation-promoting genes by
the actions of Polycomb family
(PcG) proteins for modifying
chromosomal structures. (To see
this illustration in color the
reader is referred to the web
version of this article at www
1808 HAN AND YOON
Mouse and human ES cells exhibit distinct growth char-
Doubling rates for human ES cells are charac-
teristically longer (30 to 40h) (139) than those for mouse ES
cells (17 to 19h) (172). Human ES cells require maintenance of
cell–cell contacts for propagation, and dissociation of human
ES cells triggers apoptosis by actomyosin hyperactivation (25,
131). Spontaneous differentiation is initiated from central cells
within human ES cell colonies (130), whereas spontaneous dif-
ferentiation of mouse ES cells occurs at the colony periphery.
Human ES cells are routinely cultured on a fibroblast cell feeder
of mouse ES cells. The growth factors capable of promoting
(FGF2), produced by the feeder cell layer, and insulin-like
growth factor (IGF), secreted by human ES cells, which set up
signals required for maintaining human ES cells have reported
that FGF2 is sufficient to support growth of these cells on Ma-
trigel, a substrate made up predominantly of laminin and col-
lagen but with additional unknown factors (203). It is likely that
extrinsic signals maintaining human ES cells will inhibit BMP
signaling to sustain proliferation without differentiation. BMP4
has been shown to regulate pluripotency negatively and to in-
duce trophoblast-like cell formation from human ES cells (204).
Activin/Nodal and FGF2 are capable of maintaining human ES
cells in the absence of feeder layers and other exogenous factors
has been proposed as inhibition of the BMP4 signaling pathway
mediators Smads 1 and 5. Activin A and FGF2 have recently
been shown to facilitate derivation and maintenance of plurip-
otent mouse epiblast-derived cell lines (16, 175).
Although culture conditions and extrinsic growth factors
that can support pluripotent cell maintenance have been de-
fined, it is poorly understood how the signaling pathways
controlled by these factors maintain the transcription factor
network required for pluripotency. In mouse ES cells, LIF ac-
tivates JAK/STAT signaling and mitogen-activated protein
kinase (MAPK) pathways. The choice between pluripotency
and differentiation is dependent on a balance between STAT3
and extracellular signal-regulated kinase (ERK) MAPK activ-
ity, respectively. In mouse ES cells, BMP4 prevents differenti-
ation through the inhibition of ERK (140) and induction of
other inhibitors of differentiation, such as inhibitor of differ-
that playimportant roles inpluripotency,includingc-Myc (20),
Nanog (22, 27, 118, 168), Eed (180), Jmjd1a (76), and GABPa,
which is required for the maintenance of Oct4 expression (74).
Neither STAT3 nor BMP4 activity is implicated in the
is required for the maintenance of pluripotency (6, 88). Sus-
tained activation of c-Myc in human ES cells induces differ-
entiation and apoptosis (166). Thus, consistent with the
differing extrinsic requirements, the intracellular signals reg-
be different, despite the conservation of the core transcription-
factor networks and functional similarity of the cells. Smith
maintained by the inhibition of ERK pathway and glycogen
synthase kinase 3 (GSK3) after the elimination of extrinsic
stimuli, suggesting that ES cells have an innate program for
self-replication. More recently, these inhibitions of ERK and
immature state that shares features of mouse ES cell state,
showing that these differences appear to reflect the embryonic
origin of mouse and human ES, ICM, and epiblast, respec-
tively, rather than species-specific difference (17, 49).
Epigenetic regulation of chromatin in iPS cells
Given that ES and somatic cells contain almost identical
genomic DNA, epigenetic regulation is one of the major in-
fluences on their differentiation potential and pluripotency.
pluripotency during repro-
gramming. Extrinsic signal-
ing pathways are required for
maintaining the pluripotency
of iPS cells as ES cells. Leuke-
mia inhibitory factor (LIF)
signaling activates Jak-Stat3
to induce target genes essen-
tial for pluripotency and also
to inhibit glycogen synthase
kinase-3b (GSK3b) to trigger
the translocation of b-catenin
to thenucleus for activation of
GSK3b also is inhibited by
Wnt signaling through the
(Dsh). PI3K signal can be ac-
(GFs) or LIF, and this PI3K
signal alsoinhibits theGSK3b function ofdegradation ofb-catenintomaintain self-renewal and pluripotency. The extracellular
signal-regulated kinase (Erk) pathway, which can also be activated by LIF, triggers the expression of differentiation-promoting
genes. For maintaining pluripotency and blocking differentiation, Erk must be inhibited by bone morphogenetic protein (BMP)
signals and transforming growth factor (TGF)-b/Activin A signals through Smad protein-complex formation. (To see this
illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
INDUCED PLURIPOTENT STEM CELLS1809
To maintain pluripotency in ES cells, differentiation-
triggering genes should be inactive. Polycomb group proteins
(PcGs) play important roles in silencing these developmental
regulators. The PcGs form multiple polycomb PRCs, the
components of which are conserved from Drosophila to hu-
mans (148). The PcG proteins function in two distinct poly-
comb repressive complexes, PRC1 and PRC2. Genome-wide
binding-site analyses have been carried out for PRC1 and
PRC2 in mouse ES cells and for PRC2 in human ES cells (13,
84). The genes regulated by the PcG proteins are co-occupied
by nucleosomes with trimethylated H3K27. These genes are
transcriptionally repressed in ES cells and are preferentially
activated when differentiation is induced. Interestingly, the
pluripotency factors Oct4, Sox2, and Nanog co-occupy a sig-
nificant fraction of the PcG protein–regulated genes when
acting as transcription factors (13, 84). Most of the transcrip-
tionally silent developmental regulators targeted by Oct4,
Sox2, and Nanog are also occupied by the PcG (10, 13, 84),
which are epigenetic regulators that facilitate maintenance of
the cell state through gene silencing. PRC2 catalyzes H3K27
methylation, an enzymatic activity required for PRC2-medi-
ated epigenetic gene silencing. H3K27 methylation is thought
to provide a binding surface for PRC1, which facilitates olig-
omerization, condensation of chromatin structure, and inhi-
bition of chromatin remodeling activity to maintain silencing.
PRC1 also contains a histone ubiquitin ligase, Ring1b, whose
activity appears likely to contribute to silencing in ES cells
(163) (Fig. 5). How the PcGs are recruited to genes encoding
developmental regulators in ES cells is not yet understood.
Some of the most conserved vertebrate sequences are associ-
ated with genes encoding developmental regulators, and
some of these may be sites for DNA-binding proteins that
recruit PcG proteins.
Recent studies demonstrated that the silent developmental
genes that are occupied by Oct4, Sox2, Nanog, and PcG pro-
teins experience an unusual form of transcriptional regulation
(44). These genes undergo transcription initiation but not
initiation apparatus is recruited to developmental gene pro-
moters, butRNApolymerase isincapableoffullytranscribing
these genes, presumably because of repression mediated by
the PcG. This explains why the silent genes encoding devel-
opmental regulators are generally organized in ‘‘bivalent’’
domains that are occupied by nucleosomes with histone
H3K4me3, which is associated with gene activity, and by
nucleosomes with histone H3K27me3, which is associated
with repression (7, 10, 44).
The presence of inactive RNA polymerase at the promoters
of genes encoding developmental regulators may explain
why these genes are especially poised for transcription acti-
vation during differentiation (13, 84). PcG complexes and
associated proteins may serve to pause RNA polymerase
machinery at key regulators of development in pluripotent
cells and in lineages in which they are not expressed. When
the cells are activated, PcGs and nucleosomes with H3K27
methylation are lost (13, 84, 117), allowing the transcription
vates core transcriptional networks, which maintain self-renewal by activating the pluripotent-related genes and by re-
pressing the differentiation-promoting genes by recruitment of polycomb repressive complex (PRC)2, which is responsible for
trimethylation of lysine 27 on histone 3 (H3K27me3), in which PRC1 is recruited to repress the target genes, during repro-
gramming. PRC2 is recruited to specific target sequences by DNA-binding protein, including transcription factors (TFs), and
histone deacetylase (HDAC)1 interacts with PRC2. The core PRC2 includes enhancer of zeste homologue 1/2 (EZH1/2),
suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). The modification of H3K27me3 by EZH1/2,
which is a H3K27 methyltransferase, in PRC2, provides a binding site recognized by PRC1, which includes B lymphoma Mo-
MLV insertion region 1 (BMI1), Ring1, polyhomeotic C (PHC), and chromobox protein homologue (CBX). For full repression
of target genes, binding of both PRC1 and PRC2 at specific sites is required. BMI1 of PRC1 and EZH2 of PRC2 interact with
DNA methyltransferase (DNMT), which initiates DNA methylation at CpG sites by converting cytosine to 5-methylcytosine
(5meC), and contributes the condensation of the chromosomal structures for blocking the transcription of differentiation-promoting
genes. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
Polycomb group protein (PcG)-mediated gene silencing during reprogramming. Nuclear reprogramming acti-
1810 HAN AND YOON
apparatus to function and transcribe these genes fully. The
mechanisms that lead to selective activation of genes encod-
ing specific developmental regulators are not yet understood
(81). Beyond the specific regulation of development-related
genes, ES cells maintain chromatin in a highly dynamic and
transcriptionally permissive state. Fewer heterochromatin
foci are detected in ES cell nuclei compared with differenti-
ated cells. Fluorescence recovery after photobleaching and
biochemical analyses reveal that ES cells, compared with
differentiated cells, have an increased fraction of loosely
bound or soluble architectural chromatin proteins, including
core and linker histones and heterochromatin protein HP1. A
hyperdynamic chromatin structure is functionally important
for pluripotency (115). The status of histone modifications
also indicates that the chromatin in ES cells is more tran-
scriptionally permissive than that in differentiated cells.
Consistent with the global dynamics of chromatin, ES cell
differentiation is associated with a decrease in global levels of
active histone marks, such as acetylated histone H3 and H4,
and an increase in repressive histone marks, histone H3 lysine
9 methylation (83, 115). Taken together, these unique epige-
netic characteristics of ES cells facilitate rapid but regulated
transcription, allowingdifferentiation down different cell-fate
pathways as needed by the organism.
MicroRNAs and pluripotency
Noncoding RNA composes a large fraction of vertebrate
transcriptomes. Although not all noncoding RNAs are func-
tional, many play important regulatory roles. MicroRNAs
(miRNAs) are small noncoding RNAs of *22 nucleotides in
length. They regulate gene expression through at least two
distinct mechanisms: degradation of target mRNA transcripts
and inhibition of mRNA translation (75). ES cells express a
unique set of miRNAs that are downregulated as ES cells
conserved between human and mouse and are clustered in
the genome (57, 165). miRNAs might play a role in the
maintenance of pluripotency in iPS cells as well as ES cells
(196). Lin28, one of the factors used to reprogram human fi-
broblasts (209), was recently shown to block processing of the
let-7 family microRNA in ES cells (124, 186). Let-7 family
members also have been implicated in the promotion of dif-
ferentiation of cancer stem cells (78, 209). Thus, Lin28 may
facilitate reprogramming by repressing let-7–induced differ-
entiation in fibroblasts. In this sense, inhibition of let-7 miR-
NAs by transfection of miRNA inhibitors, anti-sense RNAs,
increased the efficiency of reprogramming somatic cells into
iPS cells (114), whereas introducing of ES-specific miRNAs by
transfection of the mature form of miRNAs increased effi-
ciency of iPS cell generation from 0.01% to 0.5% to 0.4% to
0.7%, Recently, Wilson and colleagues (196) showed that
Lin28 is also repressed by miR-125, which is another differ-
entiated cell-specific miRNA (196).The inhibitions of both
miRNAs, let-7 and miR-125, may have additional effects on
reprogramming efficiency. Collectively, these data suggest
that miRNAs play important roles in reprogramming cells
into iPS cells as well as in maintaining pluripotency.
Other possible mechanisms
Nuclear reprogramming is a complex process that is not
fully understood. iPS cells are derived from proliferating so-
matic cell populations, and reprogramming technology ne-
cessitates DNA replication and cell division. Cell-cycle
duration of ES and iPS cells is much faster than that of dif-
ferentiated, cells mainly due to a shortened G1 phase. In
mouse embryonic fibroblasts (MEFs), the G1phase lasts 15 to
20h and temporally accounts for more than 80% of the cell
cycle. However, in both mouse and human ES cells, G1lasts 2
to 4h and temporally accounts for only 15% to 20% of the cell
cycle. This unique cell-cycle pattern is characterized by hy-
perphosphorylated RB protein, constitutively high activity of
cyclin E and A–associated kinases, and a lack of expression of
major CDK inhibitors (162). The role of a shortened G1phase
in maintaining pluripotency is not clear, although the exclu-
sivity of it among cells that are pluripotent suggests that it is
switches to an MEF-like pattern (147). Another difference
between ES cells and somatic cells is the high level of telo-
merase activity in ES and many adult stem cells.
Similar to ES cells, iPS cells exhibit a cell cycle with a
shortened G1phase (103) and elevated telomerase activity
(171, 172, 211). During reprogramming, fibroblasts not only
become pluripotent, but they also become immortal. Fibro-
blasts proliferate for a finite period before entering into se-
nescence. In contrast, ES cells and iPS cells do not experience
such a limitation. Immortalization requires that at least two
barriers be overcome: cellular senescence and telomere
shortening (31, 53). Rb and p53 are the key senescence-
inducing factors. In ES cells, the Rb pathway is constitutively
inactivated because of hyperphosphorylation (147), whereas
certain aspects of p53 function are compromised (141). In-
hibition of p53 function and Ink4/Arf promotes the repro-
gramming of somatic cells to pluripotent cells (56, 68, 87, 105,
Cells can enter a so-called replication crisis state in which
they undergo apoptosis if their telomere erodes below a crit-
of telomerase must be upregulated. c-Myc directly upregu-
lates the transcription of Tert, the gene encoding the enzy-
matic subunit of the telomerase (199). It is unclear whether
elevated telomerase activity in iPS cells is due to ectopic ex-
pression of c-Myc and how much the resulting change of tel-
omerase activity contributes to reprogramming. It is also
unclear how the four factors find ways to inactivate Rb and
p53 and to what extent to 11% (4,101). Mouse fibroblasts
dedifferentiated in vitro from animals madefrom iPS cells that
were established by doxycycline-inducible vectors showed
that the efficiency of reprogramming is approximately 2% to
4%, compared with approximately 0.05% in direct repro-
gramming from fibroblasts (190, 192). One group reported
that treatment with VPA increased the reprogramming effi-
ciency up to 11% for mouse fibroblasts (59). However, a dif-
ferent group observed reduced reprogramming when they
used the piggyBac transposon with VPA treatment (212). The
lack of standardization of characterizing iPS cell formation
makes comparing reprogramming efficiencies between dif-
ferent laboratories difficult (101). Specific types of somatic
cells, such as keratinocytes, stomach cells, and liver cells, are
the cell status, which might be amenable to change. The sys-
tem of doxycycline-inducible iPS cell generation is useful for
measuring the efficiency and kinetics of reprogramming in
INDUCED PLURIPOTENT STEM CELLS1811
murine fibroblasts (14, 159, 190). By withdrawal of doxycy-
cline after various periods, it was shown that transgene ex-
pression was essential for a minimum of 10 to 12 days to
initiate cellular reprogramming. Longer exposure to doxycy-
cline resulted in an increased number of reprogrammed cells
(14, 160). This system revealed the kinetics of pluripotency
marker appearance during reprogramming (14, 159). The
expression of alkaline phosphatase, which is a key marker for
pluripotency, is followed by Oct4 expression. The stochastic
nature of reprogramming could be suggested by the activa-
tion of endogenous Oct4-driven or Nanog-driven reporter
genes at different times (113). An interesting recent experi-
ment used ‘‘secondary’’ iPS cells from mice derived from
‘‘primary’’ iPS cells with drug-inducible provirus. The segre-
gation of each factor by germline transmission was identifi-
able and showed that a single copy of each of the four factors
is sufficient to allow optimal reprogramming frequency and
kinetics (106). Furthermore, by using the introduction of
complementary factors into cell lines carrying single copies of
each factor, it has been shown that Klf4 and c-Myc act earlier
during reprogramming, conceivably by inducing epigenetic
alterations that assist the binding of Oct4 and Sox2 to their
target genes (106). These conclusions were confirmed and
extended by Plath and colleagues (158), showing that the ec-
topic expression of c-Myc is necessary only during the initial
stages of reprogramming and can be substituted by VPA. c-
Myc or VPA might enhance the interaction of Oct4, Sox2, and
Klf4 with their target genes to repress somatic cell-specific
gene expression and initiate the pluripotency-related gene
(122, 158, 190).
Two recent studies demonstrated that molecular markers
are sequentially expressed during reprogramming of mouse
fibroblasts (14, 160). In the first 3 to 5 days after viral trans-
duction, the fibroblast-specific marker THY-1 was down-
phosphatase (AP) was upregulated (14) in a large proportion
of fibroblasts. In subsequent days, a population of SSEA-1–
positive cells emerged within the previously THY-1–negative
orAP-positive cells.Around 10to14daysafter theinitial viral
transduction, the endogenous Oct4 or Nanog locus was re-
activated in a small percentage of cells within the SSEA-1–
positive population. Fully reprogrammed iPS clones that are
independent of ectopically expressed factors can be isolated
only at this stage.
Clinical Applications of iPS cells
iPS cells, as well as ES cells, can be used as the pluripotent
starting material for differentiated cells or tissues in regener-
ative medicine (85, 120). In a rodent model of Parkinson
disease, Wernig and colleagues (193) demonstrated that do-
paminergic neurons differentiated from mouse iPS cells could
integrate into the host brain and improve symptoms of rats
with Parkinson disease. In another study, iPS cells derived
from a tail-tip fibroblast of a humanized sickle cell anemia
mouse model were generated, and the sickle hemoglobin al-
lele was corrected by introducing the intact human wild-type
b-globin gene by homologous recombination (51). These
gene-corrected iPS cells provided the basis for the derivation
of hematopoietic stem and progenitor cells. Transplantation
of gene-corrected hematopoietic stem and progenitor cells
into sublethally irradiated mice resulted in robust engraft-
ment and a significant reduction of the sickle cell hemoglobin
at 4 and 8 weeks after transplantation. Hemoglobin levels, red
cell morphology, and clinical indices of the animals were
significantly improved (51).
Although ES cells have pluripotent potential for differen-
tiating into any tissues, ethical considerations delay the es-
tablishment of new human ES cells, as we have to destroy
early human embryos for the generation of human ES cells,
and ES cells do not display the autologous genotypes of pa-
tients (38). Human ES cells, however, were recently approved
a human clinical trial. A biotechnology company obtained
FDA approval for a phase 1 clinical trial of human ES cell–
derived oligodendroglial progenitor cells in subacute thoracic
spinal cord injuries (215). Another company received U.S.
FDA permission to test its spinal cord stem cells in 12 patients
with amyotrophic lateral sclerosis (110, 111, 185).
Human iPS cells provide a suitable system for regenerative
possessing potency similar to that of human ES cells (205).
Now that iPS cells can be derived from the patient, human iPS
cells might be an ideal cell source for cell therapy. However,
numerous challenges remain before iPS cells might be con-
sidered for patient-specific therapy: the use of retroviral vec-
tors to introduce reprogramming factors into cells; the use of
the oncogene c-Myc to achieve reprogramming; and the in-
tegration of retroviral vectors into the genome. These ma-
nipulations genetically modify the starting cells, and
genetically modified cells face significant regulatory hurdles
for therapeutic applications. Human iPS cell lines may show
significant differences in differentiation potential for specific
lineages, as do human ES cells (135). The effects of patient
disease on the differentiation potential of patient-derived iPS
cells should be addressed.
Another consideration for ensuring the therapeutic effec-
tiveness of cell therapy, regardless of the cell source, will be
the environment in which the cells are placed. Cell therapy
such as type 1 diabetes, because the transplanted cells may be
destroyed by the host immune response. Therefore, addi-
tional treatments, such as immunosuppressive treatments,
might be required. The replaced neural-lineage cells in spinal-
cord injuryorimplanted cardiomyocytesinheartdiseasemay
provoke inflammation in the injured spinal cord or damaged
heart. In addition, the ischemic environment of the infarcted
heart that lacks a normal vascular network and perfusion is a
limiting factor for implanted cardiomyocyte survival. Given
these complicated microenvironmental changes happening
between implanted cells and the diseased host tissues, the
development and implementation of iPS-cell–based therapies
can be far more complex than simple differentiation into re-
placement cells. As seen in the use of monoclonal antibodies
and the use of viral vectors for gene therapy, novel thera-
peutics often require many years of development before they
come to fruition and deliver results in clinical trials.
iPS cells generated by manipulating pluripotency-related
transcription factors shed new light on human regenerative
medicine. Although they are limited now in number, a rapid
increase of reports outlining potential therapeutic uses of iPS
1812HAN AND YOON
cells can be expected in the near future. Although it is very
promising in early preclinical models, this technology will
undoubtedly require further refinement before clinical ap-
plication will become feasible. To avoid potential drawbacks
in clinical applications, such as tumorigenesis of iPS cells
generated by Yamanaka’s original methods by using retro-
viral vectors, alternative strategies to generate iPS cells with
fewer or absent genome modifications have been successfully
developed in a very short period. The potential to create
disease- and patient-specific pluripotent cells from fibroblasts
or other types of somatic cells of any donor makes it possible
to imagine personalized medicine without any immunologic
complications. The ability to generate pluripotent cells that
differentiate into most cell types will also make possible the
generation of in vitro models of human disease and drug-
screening tools (29, 137, 193). Several human disease models
have already been established by using patient-specific iPS
cells from patients with Parkinson disease (156), thalassemia
(189), Lou Gehrig disease (29), spinal muscular atrophy (33),
and familial dysautonomia (82). Moreover, given genetic
disorders can be corrected by homologous recombination to
restore the functions of affected tissues. This correcting of
genetic defects by using iPS cell technology has already been
accomplished with Fanconi anemia patient–specific iPS cells
(142). iPStechnologies now makeit possible tocreate celllines
abilities to detoxify and metabolize drugs, suitable for use in
the pharmaceutical industry for new drug development.
Although still much remains to be understood about the
detail and mechanisms of nuclear reprogramming, and many
challenges must be overcome to generate safer iPS cells, the
tremendous potential of iPS cell-generating technologies in
research and clinical applications will lead to and expand to
incurable diseases. Regenerative medicine is becoming more
realistic and closer to clinics by fascinating and emerging new
nuclear reprogramming technologies.
This work was supported in part by NIH grants
(RO1HL084471, R21HL097353, RC1 GM092035-01, HHSN
268201000043C), and PHS grant (UL1 RR025008) from the
Clinical and Translational Science Award program, NIH,
NCRR, and a grant (SC4300) from the Stem Cell Research
Center of the 21st Century Frontier Research Program funded
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Address correspondence to:
Dr. Young-sup Yoon
Director of Stem Cell Biology
Associate Professor of Medicine
Department of Medicine
Division of Cardiology
Emory University School of Medicine
1639 Pierce Drive, WMB 3009
Atlanta, GA 30322
Date of first submission to ARS Central, November 30, 2010;
date of acceptance, January 1, 2011.
BMP¼bone morphogenetic protein
ERK¼extracellular signal regulated kinase
FGF¼fibroblast growth factor
GSK¼glycogen synthase kinase
ICM¼inner cell mass
iPS¼induced pluripotent stem
LIF¼leukemia inhibitory factor
MAPK¼mitogen-activated protein kinase
MEF¼mouse embryonic fibroblast
PcG¼polycomb group protein
PRC¼polycomb repressive complex
RIS¼retroviral integration site
SCNT¼somatic cell nuclear transfer
1820HAN AND YOON