The cloning of animals from adult cells has demonstrated that
the developmental state of adult cells can be reprogrammed
into that of embryonic cells by uncharacterized factors within
the oocyte. More recently, transcription factors have been
identified that can induce pluripotency in somatic cells without
the use of oocytes, generating induced pluripotent stem (iPS)
cells. iPS cells provide a unique platform to dissect the molecular
mechanisms that underlie epigenetic reprogramming.
Moreover, iPS cells can teach us about principles of normal
development and disease, and might ultimately facilitate the
treatment of patients by custom-tailored cell therapy.
Mammalian development is a unidirectional process during which
there is a progressive loss of developmental potential. It begins with
the formation of a unicellular zygote and ends with the establishment
of the 220 specialized cell types of the mammalian body. According
to their decreasing differentiation potential, specific terms have been
assigned to the individual cell populations that arise during
development, including totipotency, pluripotency, multipotency and
unipotency (Fig. 1, see also Glossary inBox 1). Each cell population
is thought to have a characteristic epigenetic pattern that correlates
with its differentiation potential (Fig. 1). As shown in Fig. 1 (which
is adapted from C. H. Waddington’s ‘epigenetic landscape’ model)
(Waddington, 1957), a marble rolling down a hill into one of several
valleys illustrates the declining developmental potential of individual
cell populations. At each bifurcation point, the potential of the
marble (cell) to choose different routes (cell fates) diminishes.
Under certain experimental conditions, differentiated cells can
revert into a less differentiated state, a process termed ‘nuclear
reprogramming’ (Box 2). Examples include the generation of
pluripotent embryonic stem (ES) cells from unipotent B
lymphocytes or neurons by somatic cell nuclear transfer (SCNT)
(Eggan et al., 2004; Hochedlinger and Jaenisch, 2002a; Li et al.,
2004), or the derivation of pluripotent embryonic germ (EG) cells
from unipotent primordial germ cells (PGCs) upon cell explantation
(Matsui et al., 1992; Resnick et al., 1992). Reprogramming also
describes the conversion of one differentiated cell type into another,
for instance of a B lymphocyte into a macrophage (Xie et al., 2004),
or a fibroblast into a muscle cell (Davis et al., 1987), following the
expression of a single transcription factor (Fig. 1). Because these two
examples of cell fate change may not involve a gain in differentiation
potential, the term ‘lineage conversion’ or ‘transdifferentiation’ is
currently used to describe them (Box 2). In Fig. 1, such cell fate
changes are illustrated by the marble moving uphill
Development 136, 509-523 (2009) doi:10.1242/dev.020867
Epigenetic reprogramming and induced pluripotency
Konrad Hochedlinger1and Kathrin Plath2
1Massachusetts General Hospital Cancer Center and Center for Regenerative
Medicine, Department of Stem Cell and Regenerative Biology, Harvard Stem Cell
Institute, Harvard University, 185 Cambridge Street, Boston, MA 02114, USA.
2University of California Los Angeles, David Geffen School of Medicine, Department
of Biological Chemistry, Jonsson Comprehensive Cancer Center, Molecular Biology
Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell
Research, 615 Charles E. Young Drive South, BSRB 390D, Los Angeles, CA 90024,
e-mails: email@example.com; firstname.lastname@example.org
Box 1. Glossary of terms
Ability of a cell to give rise to all cells of an organism, including
embryonic and extraembryonic tissues. Zygotes are totipotent.
Ability of a cell to give rise to all cells of the embryo. Cells of the inner
cell mass (ICM; see below) and its derivative, embryonic stem (ES)
cells, are pluripotent.
Ability of a cell to give rise to different cell types of a given cell
lineage. These cells include most adult stem cells, such as gut stem
cells, skin stem cells, hematopoietic stem cells and neural stem cells.
Capacity of a cell to sustain only one cell type or cell lineage.
Examples are terminally differentiated cells, certain adult stem cells
(testis stem cells) and committed progenitors (erythroblasts).
Inner cell mass (ICM)
Cells of the blastocyst embryo that appear transiently during
development and give rise to the three germ layers of the developing
Embryonic stem (ES) cells
Pluripotent cell line derived from the ICM upon explantation in
culture, which can differentiate in vitro into many different lineages
and cell types, and, upon injection into blastocysts, can give rise to
all tissues including the germline.
Primordial germ cells (PGCs)
PGCs give rise to oocytes and sperm in vivo and to embryonic germ
(EG) cells when explanted in vitro.
Embryonic germ (EG) cells
Pluripotent cell line derived from explanted PGCs. In contrast to
pluripotent ICM and ES cells, PGCs are unipotent but become
pluripotent upon explantation in culture.
Embryonic carcinoma (EC) cells
Pluripotent cell line originating from transformed PGCs. EC cells are
derived from teratocarcinomas.
Germline stem (GS) cells
Unipotent cell line derived from mouse testes, which reconstitutes
spermatogenesis when transplanted into sterile recipients.
Multipotent germline stem (mGS) cells
Pluripotent stem cell line derived from GS cells. mGS cells cannot
reconstitute spermatogenesis, but have gained the potential to
produce teratomas and chimeric animals.
Induced pluripotent stem (iPS) cells
Cells generated by the overexpression of specific transcription factors
in mouse or human somatic cells, which are molecularly and
functionally highly similar to ES cell counterparts.
Insertion of a viral genome near endogenous genes, resulting in gene
activation or silencing. Retrovirus-mediated insertional mutagenesis
in hematopoietic cells can enhance self-renewal in vitro and cause
cancer in vivo.
(dedifferentiation) or across valleys (lineage conversion). In addition
to experimentally induced changes of cell fate, the term
reprogramming is also associated with the molecular changes that
occur during normal germ cell development (Surani et al., 2007),
which are not accompanied by a change in cell fate (Box 2).
Studies on the different forms of reprogramming have yielded
important insights into the molecular mechanisms of normal
development and disease, such as olfactory receptor choice (Eggan
et al., 2004; Li et al., 2004) and cancer (Blelloch et al., 2004;
Hochedlinger et al., 2004; Li et al., 2003), and have recently led to
the identification of a defined set of transcription factors that can
convert a mature cell into a pluripotent state in the culture dish,
creating induced pluripotent stem (iPS) cells (Takahashi and
Yamanaka, 2006). Reprogramming to pluripotency with defined
factors enables the efficient derivation of patient-specific,
autologous stem cells that have considerable potential in the study
and treatment of human diseases. Indeed, skin cells reprogrammed
to pluripotency with the ‘Yamanaka’ transcription factors have
recently been shown to alleviate the symptoms of Parkinson’s
disease (Wernig et al., 2008c) and sickle cell anemia (Hanna et al.,
2007) in mouse models. In addition to cellular therapy, patient-
specific iPS cells may enable the establishment of in vitro models of
complex genetic diseases, which are not yet well understood at the
molecular level. To this end, patient-specific iPS cell lines have been
derived from individuals with a variety of diseases, including
diabetes, Parkinson’s disease and Amyotrophic Lateral Sclerosis
(ALS) (Dimos et al., 2008; Park et al., 2008a); these cell lines are
expected to facilitate the in vitro identification of novel drugs for the
treatment of these disorders.
In this manuscript, we set recent advances in the induction of
pluripotency in somatic cells with transcription factors into historical
context and discuss the mechanisms that underlie this process in
relation to alternative routes to reprogramming cell fate, including
routes that are induced experimentally or that occur during normal
Scientific milestones leading to reprogramming
with defined factors
Nuclear transfer (NT)was developed to assess whether the nuclei of
differentiated cells remain equivalent to the nuclei of embryonic
cells,and constituted the first attempts to reprogram an adult cell into
a pluripotent embryonic state. Seminal experiments (see Box 3 for
milestones in nuclear reprogramming) in amphibians in the 1950s
and 1960s (Briggs and King, 1952; Gurdon, 1962), and later in
mammals (Wilmut et al., 1997), demonstrated that the genomes of
individual adult cells, and even those of terminally differentiated
cells (Eggan et al., 2004; Hochedlinger and Jaenisch, 2002a; Inoue
et al., 2005; Li et al., 2004), remain able to generate viable cloned
animals, indicating that the developmental restrictions imposed on
the genome during differentiation must be due to reversible
epigenetic modifications, rather than to permanent genetic changes
(Hochedlinger and Jaenisch, 2002b). These studies also indicated
that the oocyte must contain factors that mediate the reprogramming
of adult cells into an embryonic state; factors that should be
identifiable and that could be used to induce pluripotency when
expressed in somatic cells.
The derivation and stable maintenance of pluripotent cell lines
was also instrumental for the reprogramming of somatic cells to
pluripotency in vitro (see Box 3). Specifically, the study of
teratocarcinomas in the 1950s and 1960s (Stevens, 1967; Stevens
and Little, 1954) led to the isolation of pluripotent embryonal
carcinoma (EC) cell lines from teratocarcinomas (Finch and
Ephrussi, 1967; Kahan and Ephrussi, 1970; Kleinsmith and Pierce,
1964), and subsequently to the derivation of ES cells from
Development 136 (4)
Muscle B cell MacrophageFibroblast
Repression of lineage-specific
genes by Polycomb proteins;
ICM/ES cells, EG cells,
EC cells, mGS cells
Only active X chromosomes;
Global repression of differentiation
genes by Polycomb proteins;
Global DNA demethylation
Adult stem cells
Fig. 1. The developmental potential and
epigenetic states of cells at different stages of
development. A modification of C. H.
Waddington’s epigenetic landscape model, showing
cell populations with different developmental
potentials (left) and their respective epigenetic states
(right). Developmental restrictions can be illustrated
as marbles rolling down a landscape into one of
several valleys (cell fates). Colored marbles
correspond to different differentiation states (purple,
totipotent; blue, pluripotent; red, multipotent;
green, unipotent). Examples of reprogramming
processes are shown by dashed arrows. Adapted,
with permission, from Waddington (Waddington,
Box 2. Definitions of nuclear reprogramming
The term nuclear reprogramming is used to describe either
functional or molecular changes to cells undergoing fate changes.
When used as a functional term, reprogramming describes
experimentally induced, stable changes in cell fate. It is most often
used in the context of the reprogramming of adult cells into
pluripotent cells, which can be achieved in various ways; for
example, by somatic cell nuclear transfer (SCNT), by the fusion of
somatic cells with pluripotent cells, by explanting germline cells (see
Glossary, Box 1), or by the expression of a defined set of transcription
factors in somatic cells (for a review, see Jaenisch and Young, 2008).
Functional reprogramming also includes the stable conversion of one
differentiated cell type into another by transcription factors; for
example, the conversion of B cells into macrophages, fibroblasts into
muscle cells or pancreatic acinar cells into β cells. The terms
‘transdifferentiation’ and ‘lineage conversion’ are used to describe
this latter type of reprogramming because it is unclear if it involves
the de-differentiation of cells into a less-differentiated progenitor cell,
as it occurs during the reprogramming of adult cells into pluripotent
cells. When used as a molecular term, reprogramming describes the
molecular changes that cells undergo as their fate changes. For
example, during the epigenetic reprogramming of cells, the
promoter regions of pluripotency genes undergo demethylation
following either SCNT or induced pluripotency. Epigenetic
reprogramming has also been used to describe certain molecular
changes that occur during development, irrespective of changes to
the differentiation state of cells, such as the DNA and histone
methylation changes that occur during germ cell maturation.
Development 136 (4)
blastocysts (Evans and Kaufman, 1981; Martin, 1981) and of
embryonic germ (EG) cells from primordial germ cells (PGCs)
(Matsui et al., 1992; Resnick et al., 1992) (Box 1). While these cell
lines remain undifferentiated and immortal in culture, they undergo
differentiation into all cell types when reintroduced into blastocysts
(Bradley et al., 1984; Brinster, 1974; Matsui et al., 1992; Mintz and
Another crucial observation for reprogramming studies was
that EC, ES and EG cells can reprogram other somatic cells when
fused with them, generating pluripotent tetraploid hybrid cells
(Cowan et al., 2005; Miller and Ruddle, 1976; Tada et al., 1997;
Tada et al., 2001). These experiments indicated that pluripotent
cells also harbor reprogramming activity and that the pluripotent
state is dominant over the differentiated state (Box 3). Further
investigations indicated that nuclear factors are responsible for
reprogramming by cell fusion (Do and Scholer, 2004), as well as
by NT (Egli et al., 2007), and hence suggested that transcription
factors might be involved. Indeed, more recent work has indicated
that the overexpression of the transcription factor Nanog, which
is required for the establishment of ES cells (Chambers et al.,
2003; Mitsui et al., 2003), enhances the formation of
reprogrammed cell hybrids by up to 200-fold (Silva et al., 2006).
However, the underlying mechanisms remain elusive.
Previous work had already indicated that individual
transcription factors, when overexpressed or deleted, could induce
cell fate changes in somatic cells. For example, experiments by
Harold Weintraub and colleagues showed that the overexpression
of the myogenic transcription factor Myod was sufficient to
convert fibroblasts into myogenic cells (Davis et al., 1987) (Fig.
2). Similarly, the elimination of Pax5 from mouse B cells results
in their dedifferentiation into progenitors that can give rise to
multiple hematopoietic lineages (Nutt et al., 1999), and the
overexpression of the transcription factor Cebpα (CCAAT
enhancer binding protein alpha) can reprogram mouse B and T
cells into macrophages (Laiosa et al., 2006; Xie et al., 2004) (Fig.
2). Similar to the transforming effects of transcription factors in
adult cell lineages, which result in lineage conversion, the
perturbation of embryonic transcription factors can induce major
cell fate changes in embryonic cells (Fig. 2). For instance, the
ectopic expression of the transcription factor Cdx2 in ES cells
results in the formation of trophectodermal stem cells from ES
cells (Niwa et al., 2005). Likewise, the ectopic expression of Gata
factors induces the formation of primitive endoderm (Fujikura et
al., 2002; Shimosato et al., 2007). Together, these data indicated
that the overexpression of individual transcription factors in
closely related cells could induce stable cell fate changes. These
observations provided the rationale for subsequent attempts to
reprogram cell types beyond the boundaries of their cell lineage
and differentiation state, including attempts to induce
pluripotency in differentiated cells through the overexpression of
Generation of iPS cells from mouse and human
Kazutoshi Takahashi and Shinya Yamanaka extended the
observations that ES cells contain dominant reprogramming
activity and that transcription factors are potent inducers of cell fate
changes by identifying four transcription factors, Oct4, Sox2, Klf4
and cMyc, from 24 predominantly ES cell-specific genes, that were
sufficient to reprogram adult mouse cells (fibroblasts) into ES-like
iPS cells when expressed retrovirally (Fig. 3) (Takahashi et al.,
2007). Initially, reprogrammed cells were identified based on drug
selection for the expression of the ES cell-specific, but non-
essential, gene Fbx15. These first-generation iPS cells were similar,
but not identical, to ES cells (Fig. 3). For example, their
transcriptional and epigenetic patterns appeared to be only partially
reset from the fibroblast to the ES cell state. Moreover, these cells
did not support the development of viable chimeric mice upon
injection into blastocysts, which is indicative of a partially
B cells Fibroblasts
? ? cell
Transcription factor-induced reprogramming
+/– Factor Ectopic expression/deletion of transcription factor
+Pdx1, Ngn3, Mafa
Fig. 2. Examples of transcription factor-mediated
reprogramming. Hierarchy of cell populations (blue shading) that
appear during normal development and their relationship to each other
(green lines). Dashed red lines illustrate examples of transcription factor-
induced reprogramming. The bracketed cMyc gene indicates that this
factor is dispensable for reprogramming. ICM, inner cell mass; ES,
embryonic stem cell; iPS, induced pluripotent stem cell.
Box 3. Key milestones that led to the reprogramming
of somatic cells into iPS cells by transcription factors
1952 First nuclear transfer experiments with frogs (Briggs and King,
1962 Cloned tadpoles generated from frog intestinal cells (Gurdon,
1964 Demonstration that single, teratoma-derived embryonic
carcinoma (EC) cells are pluripotent (Kleinsmith and Pierce,
1976 Demonstration that EC cells can reprogram somatic cells in
hybrids (Miller and Ruddle, 1976).
1981 Isolation of mouse embryonic stem (ES) cells from blastocysts
(Evans and Kaufman, 1981; Martin, 1981).
1987 Reprogramming of fibroblasts into muscle cells by Myod
(Davis et al., 1987).
1992 Isolation of mouse embryonic germ (EG) cells from fetal germ
cells (Resnick et al., 1992; Matsui et al., 1992).
1997 First animal cloned from an adult cell (Dolly) (Wilmut et al.,
1998 Derivation of human ES cells (Thomson et al., 1998).
2004 Reprogramming of B cells into macrophages by Cebpα (Xie
et al., 2004).
2006 First induced pluripotent stem (iPS) cells generated from adult
mouse fibroblasts (Takahashi and Yamanaka, 2006).
Subsequent studies, however, showed that the identification of
iPS cells based on drug selection using the promoters of the essential
pluripotency genes Oct4 or Nanog (Maherali et al., 2007; Okita et
al., 2007; Wernig et al., 2007) gives rise to cells that are more similar
to ES cells. The Oct4 and Nanog genes may become reactivated
more selectively in cells undergoing faithful reprogramming,
whereas Fbx15 may be reactivated more broadly in treated cells,
thus enriching for partially reprogrammed cells. Another important
observation was that a delay in drug selection yields more faithfully
reprogrammed iPS colonies (Maherali et al., 2007; Okita et al.,
2007; Wernig et al., 2007). This led to experiments showing that
morphological criteria alone are sufficient to obtain iPS cells
(Blelloch et al., 2007; Maherali et al., 2007; Meissner et al., 2007).
At the molecular level, completely reprogrammed iPS cells show
transcriptional patterns that are highly similar to those in ES cells,
as well as DNA demethylation of the promoter regions of Oct4 and
Nanogand,in female cells,the reactivation of the somatically silent
X chromosome. Moreover, iPS cells exhibit global patterns of
histone methylation, including histones H3 lysine 4 (K4) and lysine
27 (K27) trimethylation, that are virtually indistinguishable from
those in ES cells (Maherali et al., 2007; Mikkelsen et al., 2008; Okita
et al., 2007; Wernig et al., 2007) (Fig. 3). At the functional level,
completely reprogrammed iPS cells produce viable chimeric mice
and contribute to the germline, and even support the development of
embryos that are derived entirely from iPS cells (Hanna et al., 2008;
Kim et al., 2008b; Wernig et al., 2007).
Since the 2006 landmark paper by Kazutoshi Takahashi and
Shinya Yamanaka, iPS cell have been generated from the cells of
multiple tissues, including blood (Hanna et al., 2008), liver (Aoi et
al., 2008), stomach (Aoi et al., 2008), pancreas (Stadtfeld et al.,
2008a), brain (Eminli et al., 2008; Kim et al., 2008b; Shi et al.,
2008b), intestine and adrenals (Wernig et al., 2008a) (Table 1).
Moreover, human fibroblasts (Lowry et al., 2008; Park et al., 2008b;
Takahashi et al., 2007) and keratinocytes (Aasen et al., 2008;
Maherali et al., 2008) have been converted into iPS cells using the
same, and also using a different combination of factors, including
OCT4, SOX2, LIN28 and NANOG (Yu et al., 2007). These results
suggest that reprogramming to pluripotency with defined
transcription factors is a process that can be induced in many cell
types derived from all three germ layers.
Intermediate stages of reprogramming
Transcription factor-induced reprogramming to pluripotency is a
gradual process; it takes 1-2 weeks to generate iPS cells from
mouse fibroblasts (Brambrink et al., 2008; Stadtfeld et al., 2008b).
To dissect the mechanism of reprogramming, it has been
informative to study partially reprogrammed cells (Fig. 3), such as
cells generated by the method of Fbx15 selection that was initially
used to identify iPS cells (Takahashi and Yamanaka, 2006).
Partially reprogrammed cells are also frequently obtained when
morphological criteria alone are employed to isolate iPS cells
(Mikkelsen et al., 2008). In partially reprogrammed cells lines, the
retroviral transgenes that are generally used to deliver the various
reprogramming factors are not silenced, and the endogenous
pluripotency genes show incomplete demethylation and
reactivation (Mikkelsen et al., 2008; Takahashi and Yamanaka,
2006) (Fig. 3). Genome-wide expression analyses have shown that
partially reprogrammed cell lines derived from B cells and
fibroblasts are more similar to each other than to their cells of
origin, suggesting that there could be one or several common
intermediate states in which somatic cells get trapped in the culture
dish, irrespective of the cell of origin (see Fig. 1).
Interestingly, partially reprogrammed cell lines show the
activation of lineage-specific genes that are not normally expressed
in the starting cell population or in pluripotent cells, such as Gata6,
Sox9 and Pax7 (Mikkelsen et al., 2008). Consistent with the notion
that the ectopic expression of these lineage-specific transcription
factors might prevent a cell from being converted into a pluripotent
state, the knockdown of any of these genes resulted in a more
efficient transition from the partially to a fully reprogrammed state
(Mikkelsen et al., 2008) (Fig. 3). In agreement with the finding that
the inhibition of differentiation-associated pathways is important for
inducing pluripotency, the treatment of partially reprogrammed iPS
cells with inhibitors of the extracellular signal-related kinase (ERK)
and glycogen synthase kinase 3 (GSK3)/Wnt signaling cascades
(Ying et al., 2008) facilitates their efficient conversion into fully
reprogrammed iPS cells (Silva et al., 2008). Interestingly, the
inhibition of differentiation seems to be important also during
normal germ cell development (Surani et al., 2007). In mammals,
for example, the repression of the differentiation-associated Hox
genes by the SET domain protein Blimp1 is essential for the
Development 136 (4)
Partially reprogrammed cells
(stable cell lines)
• Knockdown of lineage genes
• Inhibition of DNA methylation
• Somatic markers silenced
• Activation of SSEA1
• Silencing of retroviral transgenes
• Activation of pluripotency genes
• Activation of telomerase
• Reactivation of silent X chromosome
in female cells
• Teratomas and germline chimeras
• Viral transgenes on
• Proliferation genes activated
• Pluripotency genes silent
• Aberrant expression of lineage genes
• Teratomas, but no adult chimeras
Fig. 3. Steps involved in direct reprogramming to
pluripotency. The starting, intermediate and end stages
of reprogramming to pluripotency that can be identified
during the generation of iPS cells are shown.
‘Intermediate cells’ appear only transiently before
converting into iPS cells, whereas ‘partially
reprogrammed cells’ can be stably propagated and
converted into iPS cells upon treatment with DNA
demethylating agents and knockdown of lineage-
specific genes. Although not proven, it is assumed that
partially reprogrammed cells originate from transient
intermediate cells. The defining molecular and cellular
characteristics are shown above and below each cell
Development 136 (4)
specification of PGCs in vivo (Ohinata et al., 2005), indicating
similar principles between the maintenance of germ cell fate and the
induction of pluripotency.
While the analysis of partially reprogrammed cell states has been
informative for understanding molecular barriers to reprogramming,
a more detailed analysis of the earlier and later stages of
reprogramming is crucial for establishing the sequence of
transcriptional and epigenetic events that lead to a pluripotent state.
In attempts to define such early intermediates, two studies have
shown that the reprogramming of murine fibroblasts into iPS cells
follows a defined sequence of molecular events that begins with the
downregulation of somatic markers, such as Thy1 and collagens,
followed by the reactivation of the embryonic marker stage-specific
embryonic antigen 1 (SSEA1; Fig. 3) (Brambrink et al., 2008;
Stadtfeld et al., 2008b). SSEA1-positive cells then gradually
reactivate other markers associated with pluripotency, including
Oct4, Sox2, Nanog, telomerase (tert), and the silent X chromosome
in female fibroblasts. The reactivation of these late markers
correlates with the time window when cells become independent of
retroviral transgene expression and enter a self-sustaining
pluripotent state. It is possible that the partially reprogrammed cell
lines described above are the trapped equivalent of the transient
SSEA1-expressing cell population, although direct evidence for this
relationship is lacking (Fig. 3). The observation that the somatic
markers of a cell become downregulated before it progresses to a
pluripotent state supports the notion that the silencing of its
differentiation program is an important initial step towards re-
establishing pluripotency. It further suggests that the differentiation
state of the cell of origin for iPS cells might affect the efficiency and
kinetics of the reprogramming process.
The differentiation status of the starting cell and
NT is a very inefficient process (~1-3% of cloned blastocysts
develop into live newborns) (Hochedlinger and Jaenisch, 2006)
and the derivation of iPS cells is even less efficient, ranging from
0.01% to 0.1% (Table 2). The low efficiency of both processes has
been argued to depend on the presence of rare stem cells within
the starting population. For example, adult stem cells are present
in many tissues at about the same frequency as the success rate of
reprogramming. Some adult stem cells share transcriptional
regulators with ES cells, such as the Zinc finger protein X-linked
(Zfx) (Galan-Caridad et al., 2007) and Sox2 (Ellis et al., 2004),
and may require less epigenetic reprogramming than terminally
differentiated cells. While NT experiments have clearly
demonstrated that fully differentiated lymphocytes (Hochedlinger
and Jaenisch, 2002a; Inoue et al., 2005) and neurons (Eggan et al.,
2004; Li et al., 2004) can be reprogrammed into pluripotent ES
cells, these experiments did not exclude the possibility that adult
stem cells were the selective donors in most successful cloning
experiments. In agreement with this idea, neural stem cells
(NSCs) and keratinocyte stem cells give rise to cloned mice with
greater efficiency than do mature fibroblasts, epidermal transit
amplifying cells, or neurons (Blelloch et al., 2006; Eggan et al.,
2004; Li et al., 2007; Li et al., 2004; Wakayama and Yanagimachi,
1999). By contrast, experiments in the hematopoietic system
suggest that differentiated granulocytes are more efficient donors
for NT than are hematopoietic stem cells (Inoue et al., 2006; Sung
et al., 2006), although these experiments have recently been
challenged (Hochedlinger and Jaenisch, 2007).
Consistent with results from NT experiments is the finding that
murine NSCs can give rise to iPS cells up to 50 times more
efficiently than can mouse fibroblasts (Kim et al., 2008b). Moreover,
human keratinocytes undergo faster reprogramming than do human
fibroblasts (Aasen et al., 2008; Maherali et al., 2008), and one study
even observed an increased efficiency over fibroblasts (Aasen et al.,
2008), suggesting that keratinocytes have a transcriptional state that
is more amenable to reprogramming than that of fibroblasts or,
alternatively, that keratinocyte cultures contain more progenitors
than fibroblast cultures. It will undoubtedly be interesting to revisit
the question of whether the differentiation state of a cell affects its
reprogramming efficiency into iPS cells in a more defined system,
such as the hematopoietic lineage.
The identity of the starting cells that give rise to iPS cells remains
controversial. Two recent experiments addressed the cell-of-origin
question in different cellular systems and came to different
conclusions. In the first set of experiments, Hanna et al. attempted
to reprogram B lymphocytes into iPS cells to evaluate whether
terminally differentiated cells can give rise to iPS cells (Hanna et al.,
2008). B cells carry differentiation-associated DNA rearrangements,
which serve as unambiguous genetic markers of their differentiation
state (Hochedlinger and Jaenisch, 2002a). Interestingly, the ectopic
expression of Oct4, Sox2, cMyc and Klf4 alone was insufficient to
reprogram B lymphocytes into iPS cells, even when employing a
‘secondary’ system, in which most, if not all, cells express the four
factors homogeneously (Wernig et al., 2008a) (Table 1). The authors
had to either overexpress the transcription factor Cebpα or knock
down the transcription factor Pax5, in addition to overexpressing the
four factors, to generate iPS cells. The elimination of Pax5 has
previously been shown to endow B cells with multipotency (Nutt et
al., 1999) and the ectopic expression of Cebpα leads to the
downregulation of Pax5 and thus to the reprogramming of B cells
Table 1. Reprogramming of different mouse cell types into iPS cells
Germ layer Cell typeEfficiency Selection typeReferences
MesodermMEFs 0.01-0.1% Fbx15/Oct4/NanogTakahashi and Yamanaka, 2006; Maherali et al., 2007;
Okita et al., 2007; Wernig et al., 2007
Wernig et al., 2008
Hanna et al., 2008
Aoi et al., 2008
Aoi et al., 2008
Wernig et al., 2008
Stadtfeld et al., 2008
Kim et al., 2008; Shi et al., 2008; Eminli et al., 2008
Wernig et al., 2008
Mature B cell*,†
*Secondary cells. Differentiated cells derived from iPS cells carrying the four viral transgenes under the control of a doxycycline-inducible promoter.
†Note that mature B cells required ectopic expression of Cebpα in addition to the four reprogramming factors to produce iPS cells.
MEFs, murine embryonic fibroblasts. ND, not determined.
into macrophages (Xie et al., 2004). By contrast, progenitor B (pro-
B) cells were permissive to being reprogrammed by the four factors
alone, consistent with the notion that the differentiation state of the
starting cell might affect reprogramming efficiency.
In another set of experiments, Stadtfeld et al. used genetically
marked, terminally differentiated pancreatic β
reprogramming into iPS cells (Stadtfeld et al., 2008a). β cells gave
rise to iPS cells at a frequency comparable to that of fibroblasts (0.1-
0.2%; Table 1), demonstrating that this terminally differentiated cell
type can be reprogrammed into iPS cells by just four factors and that
adult stem cells are unlikely to be the selective cell type in successful
reprogramming experiments. There are several explanations for the
different outcomes of the reprogramming of B cells and β cells.
First, B lymphocytes belong to the mesodermal lineage, whereas β
cells are derived from endoderm; liver and stomach cells, which are
also endodermal derivatives, have recently been suggested to be
more amenable to reprogramming than fibroblasts (which are
mesodermal in origin) (Aoi et al., 2008). Alternatively, βcells could
be more easily reprogrammed than lymphocytes because the
pancreas is not organized into a hierarchical structure, as the
hematopoietic system is (which contains stem, progenitor and
differentiated cells)(Orkin and Zon, 2008), but rather reproduces its
β cell pool by self duplication (Dor et al., 2004).
Technical limitations to reprogramming
Another explanation for the low efficiency of reprogramming could
be that insertional mutagenesis, which can be caused by the
retroviruses typically used to deliver the reprogramming factors, is
potentially required for the nucleus of the starting cell to undergo
reprogramming. Previous data have shown that retroviruses can
activate endogenous genes in explanted hematopoietic stem cells,
which promoted their turnover and survival (Kustikova et al., 2005).
Similarly, one or several of the viral copies present in iPS cells might
integrate into, and activate, a gene(s) that facilitates the reacquisition
of a pluripotent, self-renewing state. However, the sequencing of
viral insertion sites in iPS cells derived from fibroblasts (Varas et al.,
2008), liver and stomach cells (Aoi et al., 2008) did not reveal any
common integration sites, suggesting that insertional mutagenesis
does not play an essential role in the induction of pluripotency. The
possibility that retroviral insertion is required for the generation of
iPS cells was finally excluded by two recent independent studies that
produced mouse iPS cells by transiently introducing the four
reprogramming factors into somatic cells using either non-
integrating adenoviruses (Stadtfeld et al., 2008c) or transient
plasmid transfection (Okita et al., 2008). The efficiency of producing
iPS cells with these transient expression methods was one to two
orders of magnitude lower than the rates achieved using retroviral
or lentiviral delivery methods, and will thus require further
optimization to be useful in research or for future therapeutic
When using retroviruses, fibroblast-derived iPS cells carry ~10-
20 proviral transgenes that express Oct4, Sox2, Klf4 and cMyc
(Maherali et al., 2007; Takahashi and Yamanaka, 2006; Wernig et
al., 2007), which are found at different copy numbers per clone,
suggesting that precise relative amounts of the individual
transcription factors are important for reprogramming. This is
consistent with observations that Oct4 and Sox2 levels in ES cells
are crucial for maintaining a self-renewing pluripotent state (Kopp
et al., 2008; Niwa et al., 2000). In further support of this idea,
reprogramming of NSCs into iPS cells in the absence of exogenous
Sox2 expression increases the overall efficiency by roughly
fourfold (Eminli et al., 2008). It is conceivable that the frequency
at which a single somatic cell receives the four viral transgenes at
the appropriate stoichiometry is extremely low, resulting in the low
overall efficiency. If viral infection is indeed the rate-limiting step,
one would predict that cells that can reactivate all four factors at the
correct stoichiometry should give rise to iPS cells at an efficiency
close to 100%. The use of iPS cells in which the four transgenes can
be reactivated with a doxycycline-inducible system (‘secondary
system’) has allowed this question to be addressed. When mouse or
human fibroblasts derived from such iPS cells were treated with
doxycycline, between 3-5% of the cells gave rise to iPS cells
(Hockemeyer et al., 2008; Maherali et al., 2008; Wernig et al.,
2008a) (Table 1). This is a 30- to 100-fold increase in efficiency
over primary cells infected directly with viruses, suggesting that
viral infection and expression are parameters that affect
reprogramming efficiency. However, these experiments also
suggest that the expression of the four factors alone is insufficient
in itself to reprogram adult cells to pluripotency and that additional
rare events must affect the overall efficiency of reprogramming. As
we discuss next, these events probably involve stochastic epigenetic
Development 136 (4)
Table 2. Comparing efficiencies between reprogramming cells to pluripotency and lineage conversion
Starting cell type(s)Product cell type TreatmentEfficiency References
Oct4, Sox2, Klf4, cMyc
Wakayama et al., 2007
Wakayama et al., 2007
Wakayama et al., 2007; Chung et al., 2006
Brook and Gardner, 1997
Durcova-Hills et al., 2006
Kanatsu-Shinohara et al., 2004
Silva et al., 2006
Silva et al., 2006
Silva et al., 2006
Hanna et al., 2008; Wernig et al., 2008;
Maherali et al., 2008; Hockemeyer et al., 2008
Xie et al., 2004
Xie et al., 2004
Laiosa et al., 2006
Davis et al., 1987
Zhou et al., 2008
Mature B cells
20%Pdx1, Ngn3, Mafa
*Secondary cells. Differentiated cells derived from iPS cells carrying the four viral transgenes under the control of a doxycycline promoter.
ICM, inner cell mass; PGC, primordial germ cell; GS cell, germline stem cell; ES cell, embronic stem cell; NSC, neural stem cell; EG cell, embryonic germ cell; mGS, multipotent
germline stem cell.
Development 136 (4)
Stochastic epigenetic events: their impact on
Several lines of evidence support the notion that stochastic
epigenetic events contribute to the low efficiency of reprogramming.
Indirect evidence for the involvement of such events comes from the
observations that reprogramming is a gradual process that takes
several weeks, and that the expression of the four factors alone is
insufficient to efficiently convert somatic cells into pluripotent cells
(see above). Moreover, a study that used reporter gene expression
has shown that the transcriptional status of genetically identical cells
can be very different. Some clonally derived daughter cells obtained
from early appearing iPS colonies carrying an Oct4-GFP reporter
reactivate GFP early, while others reactive it late, and some do not
express it at all, despite carrying identical proviral integrations
(Meissner et al., 2007). Thus, even though the cells are genetically
identical, their transcriptional pattern, and therefore their epigenetic
state, is different. Indeed, genome-wide analyses have confirmed
that striking differences exist in the transcriptional and epigenetic
signatures of partially and completely reprogrammed sibling clones
(Mikkelsen et al., 2008). Importantly, the treatment of somatic cells
or cells undergoing reprogramming with compounds that affect
chromatin modifications, including DNA and histone methylation
inhibitors (Huangfu et al., 2008a; Huangfu et al., 2008b; Mikkelsen
et al., 2008; Shi et al., 2008b), enhances the efficiency of
reprogramming significantly and facilitates the complete conversion
of partially reprogrammed cells that would otherwise fail to
reprogram into a pluripotent state (Fig. 3).
The requirement for stochastic epigenetic events to occur during
the formation of iPS cell lines might be common to other approaches
that aim to derive pluripotent cell lines from unipotent cells, given
the uniformly low efficiencies of their generation. For example, the
derivation of EG cells from PGCs (Durcova-Hills et al., 2006) and
that of multipotent germline stem (mGS) cells from germ line stem
(GS) cells (Conrad et al., 2008; Guan et al., 2006; Kanatsu-
Shinohara et al., 2004; Seandel et al., 2007) is thought to be even less
efficient, ranging from 1-3% and around 0.001%, respectively, even
though both PGCs and GS cells express many pluripotency genes,
including Oct4 and Sox2 (Table 2) (Imamura et al., 2006; Surani et
al., 2007). Similarly, the reprogramming efficiency of somatic cells
by ES cells in hybrids is less than 0.0006%,increasing to 3-4% when
Nanog is overexpressed from the ES cell genome (Silva et al., 2006)
(Table 2). These low efficiencies of reprogramming somatic cells
into pluripotent cells are in contrast to the frequencies at which ES
cell lines are generated from pluripotent blastomeres or ICM cells.
Specifically, the efficiencies of deriving ES cell lines from single
blastomeres of cleavage-stage embryos range from 50-69% for two-
cell blastomeres, 22-40% for four-cell blastomeres and 10-16% for
eight-cell blastomeres (Chung et al., 2006; Wakayama et al., 2007)
(Table 2). Moreover, it has been estimated that,on average,three out
of the ~25 ICM cells found in a blastocyst (12%) give rise to ES cell
lines (Brook and Gardner, 1997). Together, these results suggest that
undifferentiated blastomeres and ICM cells require fewer epigenetic
changes to convert into ES cell linesthan do the more differentiated
PGCs and GSCs, which require more changes and thus convert less
efficiently into pluripotent cell lines. Interestingly, the requirement
for stochastic epigenetic events might not be limited to the
derivation of pluripotent cell lines in vitro; they appear to also play
a role in normal development (Box 4).
Assuming that the stochastic events crucial for the derivation
of ES cells from ICM cells are also important for the
establishment of EG, mGS and iPS cells, one might predict that
the same genes and compounds that enhance ES cell derivation
should also facilitate the derivation of pluripotent cell types from
other sources. Indeed, the treatment of partially reprogrammed
iPS cells with inhibitors of the ERK kinase and GSK3 signalling
cascades, both of which are crucial for the derivation of ES cells
(Ying et al., 2008), results in the efficient conversion of partially
reprogrammed cell lines into fully reprogrammed iPS cells (Silva
et al., 2008). Moreover, Wnt pathway activation has beneficial
effects not only on the growth of ES cells (Sato et al., 2004), but
also for the reprogramming of somatic cells into pluripotent cells
by transcription factors (Marson et al., 2008) and for cell fusion
between somatic cells and ES cells (Lluis et al., 2008). Likewise,
loss of the tumor suppressor protein p53, which normally inhibits
the immortal growth of primary fibroblasts, enhances the
transformation of PGCs into embryonal carcinomas (Lam and
Nadeau, 2003) and increases the number of mGS cells derived
from GS cells (Kanatsu-Shinohara et al., 2004), as well as the
number of iPS colonies derived from fibroblasts (Zhao et al.,
2008), possibly by conferring immortality and/or by de-repressing
Nanog (Lin et al., 2005).
Reprogramming to pluripotency versus lineage
How does the efficiency of reprogramming adult cells into a
pluripotent state compare with the efficiency with which one
differentiated cell type converts into another? Interestingly, the direct
conversion of B cells and pre-T cells into macrophages by the
retroviral expression of Cebpα(Laiosa et al., 2006; Xie et al., 2004),
that of fibroblasts into myogenic cells by the retroviral expression
of Myod (Davis et al., 1987), and that of acinar cells into β cells by
adenoviral delivery in vivo of Pdx1, Ngn3 and Mafa (Zhou et al.,
2008) does not appear to be restrained by major epigenetic barriers,
based on the high efficiency of lineage switching (Table 2). For
example, 25-50% of fibroblasts that express Myod convert into
myogenic colonies (Davis et al., 1987). Pro- and pre-B cells that
ectopically express Cebpα transform into macrophages at a
frequency of ~65% and mature B cells at a frequency of ~35% (Xie
et al., 2004). Similarly, ~60% of pre-T cells convert into
Box 4. Transcriptional fluctuations occurring during
During normal development, stochastic fluctuations in gene
expression are thought to influence cell fate decisions. For instance,
subpopulations of clonally derived hematopoietic progenitor cells
have been found to exhibit metastable states (which persist over
multiple cell divisions), together with global changes in gene
expression and different tendencies to give rise to the erythroid or
myeloid lineages (Chang et al., 2008). The stochastic expression of
Nanog and Cdx2 during mouse preimplantation development has
also been observed and is assumed to play roles in the allocation of
the ICM and trophectoderm (Dietrich and Hiiragi, 2007). Similarly,
ES cells show fluctuations in Nanog (Chambers et al., 2007), Zinc
finger protein 42 (Zfp42 or Rex1) (Toyooka et al., 2008) and Stella
(Hayashi et al., 2008) expression, which has been correlated with
their ability to maintain pluripotency or to differentiate, respectively.
Specifically, it is assumed that ES cells that express the transcription
factor maintain pluripotency, whereas those that lose expression
become permissive for differentiation. Whether cells losing individual
pluripotency markers are poised to differentiate into the same or
different lineages remains to be determined. It is also unclear
whether the mechanisms that underlie stochastic transcriptional
changes during normal development are identical to those that occur
during the derivation of pluripotent cell lines.
macrophages upon overexpression of Cebpα (Laiosa et al., 2006).
Pancreatic acinar cells infected with adenoviruses that express Pdx1,
Ngn3 and Mafa convert into insulin-expressing β cells at a
frequency of 20% (Zhou et al., 2008). It is conceivable, therefore,
that changing cell fates within closely related cell types requires less
epigenetic remodeling, leading to the high efficiency of conversion.
Consistent with this idea, the expression of Cebpαand PU.1 in more
distantly related fibroblasts gave rise to cells that were similar, but
not identical, to macrophages that had been obtained from B cells,
and their phenotype was not maintained upon silencing of the viral
transgenes (Feng et al., 2008).
These observations raise the general question of whether
transcription factor-induced conversion across cell lineages and
germ layers can ever generate epigenetically stable cell fates that
closely mirror cell types found in vivo. It is possible that such
dramatic switches in cell identity require more extensive changes
in the epigenetic signature or involve very stable chromatin marks
that can only be reset after going through a pluripotent ground
state, i.e. they require de-differentiation and subsequent re-
differentiation. A related question, which was initially raised in
NT experiments, is whether iPS cells retain an ‘epigenetic
memory’ of their cell of origin (see Box 5).
Possible mechanisms of transcription factor-
Differential requirement for the reprogramming factors
An open question is how the reprogramming factors induce the
epigenetic changes that are associated with reprogramming to
pluripotency. Experiments in which several combinations and
orthologs of the four transcription factors were tested
demonstrated that not all four factors are required for
reprogramming (Nakagawa et al., 2008; Wernig et al., 2008b; Yu
et al., 2007). The fact that KLF4 and cMYC can be replaced by
NANOG and by the RNA-binding protein LIN28 in human
fibroblast reprogramming experiments suggests that different
molecular pathways can lead to reprogramming or, alternatively,
that these factors perform highly similar functions during this
process. In support of the latter, LIN28 was recently found to
function as a negative regulator of microRNA processing in ES
cells, specifically of members of the let-7 family (Viswanathan et
al., 2008). cMYC represses the transcription of similar miRNAs,
suggesting that LIN28 and cMYC could perturb the same
regulatory mechanisms that contribute to reprogramming (Chang
et al., 2008).
iPS colonies can be generated from mouse or human fibroblasts
in the absence of cMyc altogether, albeit at lower frequency and with
delayed kinetics (Nakagawa et al., 2008; Wernig et al., 2008b). The
effect of cMyc can be partially compensated by treating cells with
either the histone deacetylase (HDAC) inhibitor valproic acid (VPA)
or a ligand of the β-catenin pathway, Wnt3a (Huangfu et al., 2008a;
Marson et al., 2008). Moreover, VPA can replace the function of
both cMYC and KLF4 in human cell reprogramming,such that only
the expression of OCT4 and SOX2 are required to generate iPS cells
(Huangfu et al., 2008b).
The starting cell also has an effect on the requirement for
reprogramming factors. As mentioned before, NSCs, which
already express high levels of endogenous Sox2, require only the
ectopic expression of Oct4 with either Klf4 or cMyc to produce
iPS cells (Eminli et al., 2008; Kim et al., 2008b; Shi et al., 2008b),
and hepatocytes are as efficiently reprogrammed in the absence
of cMyc as in its presence (Aoi et al., 2008). Because cMyc, Klf4
and Sox2 (but not Oct4) are expressed in multiple adult tissues
and can be replaced by other orthologs during reprogramming
into iPS cells (Nakagawa et al., 2008), Oct4 appears to be the only
irreplaceable, and possibly the most important, determinant of
direct reprogramming. However, a recent study suggested that
even Oct4 might be replaceable in certain cellular contexts:
treatment of NSCs with a chemical inhibitor of the histone
methyltransferase G9a, which is responsible for silencing the
Oct4 promoter during normal differentiation (see below), gave
rise to Oct4-GFP-positive iPS cells upon ectopic expression of
Klf4 and cMyc alone (Shi et al., 2008b). When combined with the
calcium channel agonist BayK, this inhibitor even facilitated the
reprogramming of murine fibroblasts into iPS cells in the absence
of Sox2 and cMyc (Shi et al., 2008a). Although the mechanisms
by which these small compounds mediate reprogramming remain
elusive at this point, it may indeed be possible in the future to
generate iPS cells solely with chemicals.
ES cell transcription factor networks
Studies of the transcriptional circuitry that controls the pluripotent
state of ES cells might be helpful for understanding the function of
the reprogramming factors. Recent genome-wide analyses in ES
cells have suggested that the three reprogramming factors Oct4,
Sox2 and Klf4, and the transcription factor Nanog specify ES cell
identity by transcriptionally activating the self-renewal program
and by repressing lineage commitment pathways (Boyer et al.,
Development 136 (4)
Box 5. Retention of an ‘epigenetic memory’ in iPS
Does reprogramming erase all of the epigenetic modifications, the
‘epigenetic memory’, of the somatic donor cell? Interestingly, frog
embryos generated by nuclear transfer (NT) and derived from frog
somite cells retain Myod expression in cells that normally do not
express Myod, even after 24 cell divisions (Ng and Gurdon, 2008).
Similarly, cloned frog embryos derived from endoderm cells express
an endodermal marker gene in non-endodermal cells (Ng and
Gurdon, 2005). The histone variant H3.3 is deposited through
replication-independent mechanisms at actively transcribed loci, thus
marking actively transcribed genes (Henikoff and Ahmad, 2005), and
is believed to be necessary for the establishment of an epigenetic
memory, but detailed mechanisms remain unclear (Ng and Gurdon,
2008). Additional evidence from cloned frogs andmice has indicated
that animals derived from different donor cells show different
phenotypic and transcriptional abnormalities, consistent with the
retention of an epigenetic memory of the donor cell (Boiani et al.,
2002; Bortvin et al., 2003; Hochedlinger and Jaenisch, 2002b).
However, ES cells derived from blastocysts that have been cloned
from different donor cell types are indistinguishable from each other
and from fertilization-derived ES cells, based on transcriptional
profiling and their ability to give rise to normal mice (Brambrink et
al., 2006), indicating that the process of ES cell derivation selects for
faithfully reprogrammed cells. So far, no comparative analyses have
been reported on iPS cells and mice derived from different donor
cells, and genome-wide analyses of fibroblast-derived iPS cells did
not reveal any epigenetic aberrations suggestive of an epigenetic
memory (Maherali et al., 2007; Mikkelsen et al., 2008; Okita et al.,
2007; Wernig et al., 2007). This might indicate that iPS cells, like NT
ES cells, have faithfully erased any epigenetic marks present in donor
cells. However, one study showed that iPS cell-derived chimeras
derived from liver and stomach cells have increased rates of perinatal
death compared with iPS chimeras derived from fibroblasts (Aoi et
al., 2008). It will be informative to compare the epigenetic and
transcriptional patterns of iPS cells derived from a variety of donor
cell types to solve the question of whether or not a donor cell’s
epigenetic memory is retained.
Development 136 (4)
2005; Loh et al., 2006) (Fig. 4). Generally, multiple pluripotency
transcription factors co-occupy genes in ES cells that are active but
are repressed upon differentiation, and encode proteins that are
important for ES cell self-renewal and pluripotency (Fig. 4A). By
contrast, when bound by only one of these transcription factors,
target genes are often transcriptionally repressed in ES cells, and
encode regulators of lineage commitment, which become activated
upon the induction of differentiation (Fig. 4B).
Two models can explain how the association of a gene with
multiple transcription factors could increase its transcriptional
output. Protein-protein interactions between these transcription
factors could increase the affinity of the proteins for adjacent
recognition sites (Chen et al., 1998) or DNA conformations
generated by the binding of one transcription factor could favor
the binding of another (Panne et al., 2004). In support of the latter,
Oct4 binding is required for the recruitment of Smad1 and Stat3
to target sites (Chen et al., 2008). In both modes, steric constraints
on the proteins could favor the formation of unique configurations
that would create docking surfaces for co-activators required for
strong transcriptional activation (Merika et al., 1998). Indeed, the
p300 histone acetyltransferase is found at many Oct4 target sites
and is recruited to them in an Oct4-dependent manner (Chen et
al., 2008). Alterations in DNA structure induced by the binding of
multiple factors could also contribute to the specificity of their
binding (Panne et al., 2007), explaining how Oct4 and Sox2
distinguish developmentally appropriate binding sites from those
that carry the recognition motif but are irrelevant in the ES cell
It has been proposed that the four reprogramming factors do not
act on the same set of genes, as cMyc binds to many genes that are
not bound by Oct4, Sox2 or Klf4 (Chen et al., 2008; Kim et al.,
2008a). Nevertheless, cMyc shares target genes with other
transcription factors, including the family member nMyc, which can
replace cMyc in reprogramming experiments, and the cell cycle
regulator E2F1 and Zfx (Chen et al., 2008) (Fig. 4C), again
indicating cooperation among multiple transcription factors. It
remains to be shown whether cMyc does indeed have separable
functions from the other three reprogramming factors.
How do the transcription factors induce reprogramming?
Reprogramming needs to inactivate the somatic cell program and
to activate the ES cell-specific transcription programs of self-
renewal and pluripotency. One could speculate that the
reprogramming factors contribute to both functions, as they can, in
A Activating function of Oct4, Klf4 and Sox2
C cMyc function
B Repressive function of Oct4, Klf4 or Sox2
*Or binding by either
Sox2, Klf4, Nanog,
Nr0b1 or Nac1.
ES cell targets:
transcriptional regulators of the pluripotent state
Potential function during reprogramming:
activation of pluripotency regulators
ES cell targets:
metabolism and proliferation regulators
Potential function during reprogramming:
activation of the basic energy, proliferation and
metabolism program of the embryonic state
ES cell targets:
regulators of differentiation and lineage commitment
Potential function during reprogramming:
repression of somatic cell program
Fig. 4. ES cell transcription factor
network and implications for
reprogramming factors Oct4, Sox2 and
Klf4 (light blue) often co-bind promoter
regions with other transcription factors,
including Nanog, Nr0b1 (nuclear
receptor subfamily 0, group B, member
1), Esrrb (estrogen-related receptor,
beta), Zfp281 (zinc finger protein 281)
and Nac1 (nucleus accumbens associated
1; all of which have been purified in
large protein complexes with Oct4 or
Nanog), as well as with Stat3 and Smad1
(transcription factors downstream of the
Bmp4 and Lif signaling pathways that
maintain ES cell self-renewal and
pluripotency) (Chen et al., 2008; Kim et
al., 2008a; Wang et al., 2006). The
recruitment of co-activators, such as the
histone acetyltransferase (HAT) p300 is
often observed (yellow) (Chen et al.,
2008). This binding pattern is found in
transcriptionally active genes in ES cells.
ES cell target groups and implications for
reprogramming are also indicated. (B)In
ES cells, genes bound by either Oct4,
Sox2 or Klf4 are often repressed,
potentially through the recruitment of
Polycomb group (PcG) proteins or
histone deacetylases (HDACs), but
become activated upon differentiation
(Liang et al., 2008; Lee et al., 2006).
(C)cMyc is proposed to bind and activate
largely different sets of genes to Oct4,
Klf4 and Sox2, but in collaboration with
other transcription factors (Kim et al.,
2008a; Chen et al., 2008).
ES cells, be both repressive and activating. Thus, genes that encode
somatic cell regulators could be repressed by the binding of the
reprogramming factors, while self-renewal and pluripotency genes
would be turned on (Fig. 4). Autoregulatory loops, i.e. the binding
of factors to their own promoters (Boyer et al., 2005), could provide
a platform on which ectopic transcription factors can jump-start the
transcription of their endogenous counterparts to a level that is
sufficient to maintain their own expression. Furthermore, the
ectopic activation of lineage differentiation programs that has been
observed in partially reprogrammed cells might reflect a function
of Klf4 and Sox2 in normal development (Mikkelsen et al., 2008).
These transcription factors are also expressed in neural and
epidermal lineages, and could, potentially in combination with
other lineage-specific transcription factors, target genes during
reprogramming that they would not normally associate with in
The reprogramming factors could also have more global
functions that do not involve direct transcriptional control, which
remain completely unexplored. A few pleiotropic functions have
been suggested for cMyc, ranging from the control of initiation of
DNA replication (Dominguez-Sola et al., 2007) to global effects
on chromatin structure, especially on histone acetylation
(Knoepfler, 2008), which could be important for providing the
other reprogramming factors access to target sites. In agreement
with the latter observation, the inhibition of histone deacetylation
can replace cMyc in reprogramming experiments (Huangfu et al.,
2008a; Huangfu et al., 2008b). One could also envision that
ectopically expressed factors titrate proteins important for somatic
cell transcription away from the DNA and sequester them in
inactive complexes, thereby acting as differentiation antagonists,
as has been described for Myod in muscle specification (Puri et
al., 2001). Thus, reprogramming is likely to be more complex
than a simple model suggests, and will involve a number of
different mechanisms to overcome the epigenetic barriers that are
imposed during differentiation.
Overcoming epigenetic barriers
The inefficient activation of Oct4 in somatic cells following NT is
associated with the embryonic lethality of cloned mouse embryos
(Boiani et al., 2002; Bortvin et al., 2004), and Oct4 activation is a
stringent measure of reprogramming success during iPS cell
generation (Meissner et al., 2007; Wernig et al., 2007), which is in
agreement with the notion that Oct4 is absolutely required for the
establishment and maintenance of pluripotency during normal
development (Nichols et al., 1998). The Oct4 gene undergoes a
complex process of inactivation during post-implantation
development, which involves multiple layers of repression. Using
differentiating ES cells as a model system, it was shown that the
binding of transiently acting transcriptional repressors, such as
Coup-TF1/2 (chicken ovalbumin upstream promoter-transcription
factor) and Gcnf (germ cell nuclear factor) (Ben-Shushan et al.,
1995; Fuhrmann et al., 2001), leads to the recruitment of histone
deacetylases and the methyltransferase G9a, which in turn triggers
de novo methylation of the Oct4 promoter by the de novo DNA
methyltransferases Dnmt3a and Dnmt3b. Prior to de novo DNA
methylation, Oct4 can be readily reactivated when differentiating
cells are returned to ES cell culture conditions (Feldman et al.,
2006), indicating that only DNA methylation stably locks in the
repressed state. Differentiation-induced de novo DNA methylation
is not limited to Oct4 and appears to be a mechanism for the
repression of a larger set of pluripotency genes that includes Nanog,
Zfp42, Gdf3, Tdgf1, Dppa4 and Tcl1 (Deb-Rinker et al., 2005;
Farthing et al., 2008; Mohn et al., 2008), suggesting that DNA
methylation lowers the chance of these genes being inappropriately
activated upon lineage commitment.
The observation that in GS cells many pluripotency genes show
demethylation but are not expressed might explain their tendency
to spontaneously reprogram into mGS cells upon explantation
(see Glossary, Box 1) (Imamura et al., 2006; Kanatsu-Shinohara
et al., 2004; Seandel et al., 2007). Interestingly, Oct4 continues to
be highly methylated in differentiated cells that are deficient in
DNA methyltransferase 1 (DNMT1), the main enzyme that
enables the inheritance of DNA methylation patterns through
mitosis, despite the fact that many other genes become
demethylated immediately (Feldman et al., 2006). This is
presumably because of the constant recruitment of de novo
methyltransferases to the Oct4 promoter (Feldman et al., 2006).
Consistent with this observation, the reactivation of Oct4, and
probably its demethylation, occur at a very late stage of
reprogramming (Brambrink et al., 2008; Stadtfeld et al., 2008b).
However, not all pluripotency regulators are repressed through the
acquisition of DNA methylation in somatic tissues. For example,
Sox2 does not acquire this chromatin mark (Mikkelsen et al.,
2008; Mohn et al., 2008). Thus, multiple repressive mechanisms
function to silence the embryonic program, which need to be
overcome during nuclear reprogramming. In agreement with this
notion, interfering with the three repressive mechanisms that are
implicated in the silencing of Oct4 and other pluripotency genes
– histone deacetylation, histone methylation and DNA
methylation – improves the efficiency of transcription factor-
induced reprogramming (Huangfu et al., 2008a; Huangfu et al.,
2008b; Mikkelsen et al., 2008; Shi et al., 2008b). Whether these
inhibitors function by directly targeting the promoter regions of
pluripotency genes or by acting indirectly remains to be
Nucleosome packaging and a repressive chromatin structure
might ‘hide’ many of the ES cell-specific targets of the four
reprogramming factors and so prevent them from binding early in
the reprogramming process. In some instances, the reprogramming
factors could possess an intrinsic ability to alter chromatin structure
in a manner similar to that of the transcription factors HNF3
(hepatocyte nuclear factor 3) and GATA4 (GATA binding protein 4),
which bind to the albumin gene enhancer in silent chromatin and
facilitate the opening of an H1-compacted nucleosome array on this
enhancer to activate the gene (Cirillo et al., 2002). In other cases, the
reprogramming factors might act passively by blocking processes
that would normally act to maintain the repressed state of ES cell-
specific genes. We will discuss the possibilities for overcoming
DNA methylation more specifically below.
Possible mechanisms leading to DNA demethylation
The mechanisms of DNA demethylation in reprogramming and
normal development remain largely elusive. Two waves of global
demethylation can be distinguished in mammalian development.
Upon fertilization, the paternal genome becomes actively
demethylated in the zygote, while the maternal genome looses its
methylation marks passively during cleavage divisions (Jaenisch
and Bird, 2003). During the specification of PGCs, another round
of demethylation occurs in order to erase and subsequently re-
establish methylation marks associated with imprinted genes.
During reprogramming, demethylation may occur passively; that
is, the direct binding of reprogramming factors to promoter or
enhancer regions might interfere with the methylation of newly
synthesized DNA during DNA replication (Fig. 5A). This process
Development 136 (4)
Development 136 (4)
would be stochastic and would be more likely to occur if multiple
transcription factors associate with the target gene (Lin et al.,
2000). An example of the localized demethylation that would
result from such a physical hindrance of DNA methylation is seen
at the glucocorticoid receptor. This receptor can induce localized
DNA demethylation, which is required for the subsequent
recruitment of other transcription factors to neighboring sites
(Thomassin et al., 2001). Similarly, specific factors that contribute
to the stable inheritance of methylation patterns could be
stochastically impaired, such as Uhrf1 (Ubiquitin-like, containing
PHD and RING finger domains, protein), Dnmt3l, Suv39h1/2
(Suppressor of variegation 3-9 homologs), Lsh (Lymphoid-
specific helicase), as well as Piwil1/2 (piwi-like homologs 1/2;
Fig. 5A) (Bostick et al., 2007; Bourc’his and Bestor, 2006;
Kuramochi-Miyagawa et al., 2008; Lehnertz et al., 2003; Sharif
et al., 2007; Zhu et al., 2006).
Alternatively, active DNA demethylation mechanisms could be
required for the reactivation of pluripotency genes (Fig. 5B).
Compelling evidence indicates that DNA demethylation can occur
by methylcytosine-specific DNA glycosylases and by the base
excision repair machinery in plants (Choi et al., 2002; Gong et al.,
2002). A repair-based process might also function in mammals,
leading to global DNA demethylation in PGCs (Hajkova et al.,
2008). Furthermore, an active enzyme appears to be responsible
for the selective DNA demethylation of the paternal mouse
genome upon fertilization (Santos et al., 2002), and for Oct4
promoter demethylation after the transplantation of a somatic cell
nucleus into the frog oocyte (Simonsson and Gurdon, 2004).
However, the nature and existence of demethylating enzymes in
mammals remains highly controversial (Barreto et al., 2007;
Bhattacharya et al., 1999; Hendrich et al., 2001; Jin et al., 2008;
Kangaspeska et al., 2008; Metivier et al., 2008). Taken together,
how pluripotency genes become demethylated at the DNA level
– either actively or passively – is still under debate. Partially
reprogrammed cells could offer a unique system in which to
distinguish between these possibilities and to decipher the role of
other chromatin modifications.
In vivo versus in vitro reprogramming
As outlined above, the mechanisms that underlie nuclear
reprogramming by NT and transcription factors remain largely
elusive. However, both reprogramming events may involve
processes that are similar to the ones that operate during germ cell
reprogramming. PGCs erase DNA and histone methylation
patterns, as well as genomic imprints, and reactivate the X
chromosome during their development. Of note, early PGCs
express transcription factors and show a chromatin signature that
is reminiscent of that in pluripotent cells, correlating with the time
window when pluripotent EG cells can be derived (Hajkova et al.,
2008). Thus, their methylation and chromatin state must be
permissive for their spontaneous conversion into a pluripotent
state and may resemble that of nascent iPS cells. Other changes
in PGCs include active demethylation of DNA followed by the
replacement of histone variants, such as H2A.Z (Hajkova et al.,
2008). A comparison of the mechanisms that lead to DNA
demethylation and chromatin changes during PGC differentiation
and somatic cell reprogramming to pluripotency should be quite
informative, and may lead to strategies that improve the efficient
and faithful reprogramming of somatic cells by NT or by defined
A Passive demethylation
Symmetrically methylated DNAHemi-methylated DNA
B Active demethylation
Fig. 5. Pathways to DNA demethylation of key
pluripotency genes. (A)The establishment of
symmetric DNA methylation patterns could be
prevented passively during replication by the steric
hindrance of Dnmt1 due to the stochastic binding of
the reprogramming factors to target sites or by
inhibiting Dnmt1 function indirectly.
Hemimethylation of the DNA would result in a
progressive loss of methylation upon further rounds
of cell division. (B)Alternatively, DNA methylation
could be actively removed by the recruitment of a
Future explorations of the molecular mechanisms of different
reprogramming processes should shed light on two fundamental
questions in mammalian development: how is pluripotency
established and how are cell fate decisions made? It remains to be
shown if transcription factor-induced reprogramming progresses
through the intermediate stages that are normally found during
differentiation. A better understanding of transcription factor-
induced cell fate changes should improve the development, and the
efficiency, of ES cell differentiation approaches, as well as attempts
to convert one cell type directly into another. Indeed, recent work on
the transcription factor-induced reprogramming of pancreatic acinar
cells into insulin-producing βcells has been a promising conceptual
advance in this direction (Zhou et al., 2008). Solving the question of
whether any cell type can be directly derived from skin cells or if
transition through the pluripotent ground state is a requirement for
a switch across germ layers or cell lineages will surely stimulate
Reprogramming with transcription factors offers tremendous
promise for the future development of patient-specific pluripotent
cells and for studies of human diseases. The identification of
optimized protocols for the differentiation of iPS cells and ES cells
into multiple functional cell types in vitro and their proper
engraftment in vivo will be challenges in the coming years.
Moreover, the risk of oncogenic events caused by the use of potent
oncogenes and by the random integration of delivery vectors into the
genome is a major roadblock that needs to be overcome before
translating iPS cell technology into the clinic. Given that the first
small-molecule approaches aimed at activating pluripotency genes
have already been devised (Huangfu et al., 2008a; Huangfu et al.,
2008b; Marson et al., 2008; Shi et al., 2008a; Shi et al., 2008b) and
that murine iPS cells have recently been derived by using non-
integrative transient expression strategies of the reprogramming
factors (Okita et al., 2008; Stadtfeld et al., 2008c), we expect that
human iPS cells without permanent genetic alterations will soon be
We thank Raul Mostoslavsky, Thomas Graf, and members of the Hochedlinger
and Plath labs for critical reading of the manuscript. We are grateful to Jim
Resnick and Takashi Shinohara for discussion. K.P. is supported by the V and
Kimmel Scholar Foundation, an NIH Director’s Young Innovator Award and a
CIRM Young Investigator Award. K.H. is supported by an NIH Director’s
Innovator Award, the Harvard Stem Cell Institute, the Kimmel Foundation and
the V Foundation. Deposited in PMC for release after 12 months.
Aasen, T., Raya, A., Barrero, M. J., Garreta, E., Consiglio, A., Gonzalez, F.,
Vassena, R., Bilic, J., Pekarik, V., Tiscornia, G. et al. (2008). Efficient and
rapid generation of induced pluripotent stem cells from human keratinocytes.
Nat. Biotechnol. 26, 1276-1284.
Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T.
and Yamanaka, S. (2008). Generation of pluripotent stem cells from adult
mouse liver and stomach cells. Science 321, 699-702.
Barreto, G., Schafer, A., Marhold, J., Stach, D., Swaminathan, S. K., Handa,
V., Doderlein, G., Maltry, N., Wu, W., Lyko, F. et al. (2007). Gadd45a
promotes epigenetic gene activation by repair-mediated DNA demethylation.
Nature 445, 671-675.
Ben-Shushan, E., Sharir, H., Pikarsky, E. and Bergman, Y. (1995). A dynamic
balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid
receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in
embryonal carcinoma cells. Mol. Cell. Biol. 15, 1034-1048.
Bhattacharya, S. K., Ramchandani, S., Cervoni, N. and Szyf, M. (1999). A
mammalian protein with specific demethylase activity for mCpG DNA. Nature
Blelloch, R. H., Hochedlinger, K., Yamada, Y., Brennan, C., Kim, M., Mintz, B.,
Chin, L. and Jaenisch, R. (2004). Nuclear cloning of embryonal carcinoma cells.
Proc. Natl. Acad. Sci. USA 101, 13985-13990.
Blelloch, R., Wang, Z., Meissner, A., Pollard, S., Smith, A. and Jaenisch, R.
(2006). Reprogramming efficiency following somatic cell nuclear transfer is
influenced by the differentiation and methylation state of the donor nucleus.
Stem Cells 24, 2007-2013.
Blelloch, R., Venere, M., Yen, J. and Ramalho-Santos, M. (2007). Generation
of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell
Boiani, M., Eckardt, S., Scholer, H. R. and McLaughlin, K. J. (2002). Oct4
distribution and level in mouse clones: consequences for pluripotency. Genes
Dev. 16, 1209-1219.
Bortvin, A., Eggan, K., Skaletsky, H., Akutsu, H., Berry, D. L., Yanagimachi,
R., Page, D. C. and Jaenisch, R. (2003). Incomplete reactivation of Oct4-
related genes in mouse embryos cloned from somatic nuclei. Development 130,
Bortvin, A., Goodheart, M., Liao, M. and Page, D. C. (2004). Dppa3 / Pgc7 /
stella is a maternal factor and is not required for germ cell specification in mice.
BMC Dev. Biol. 4, 2.
Bostick, M., Kim, J. K., Esteve, P. O., Clark, A., Pradhan, S. and Jacobsen, S.
E. (2007). UHRF1 plays a role in maintaining DNA methylation in mammalian
cells. Science 317, 1760-1764.
Bourc’his, D. and Bestor, T. H. (2006). Origins of extreme sexual dimorphism in
genomic imprinting. Cytogenet. Genome Res. 113, 36-40.
Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P.,
Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G. et al. (2005).
Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122,
Bradley, A., Evans, M., Kaufman, M. H. and Robertson, E. (1984). Formation
of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature
Brambrink, T., Hochedlinger, K., Bell, G. and Jaenisch, R. (2006). ES cells
derived from cloned and fertilized blastocysts are transcriptionally and
functionally indistinguishable. Proc. Natl. Acad. Sci. USA 103, 933-938.
Brambrink, T., Foreman, R., Welstead, G. G., Lengner, C. J., Wernig, M., Suh,
H. and Jaenisch, R. (2008). Sequential expression of pluripotency markers
during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151-159.
Briggs, R. and King, T. J. (1952). Transplantation of living nuclei from blastula
cells into enucleated frogs’ eggs. Proc. Natl. Acad. Sci. USA 38, 455-463.
Brinster, R. L. (1974). The effect of cells transferred into the mouse blastocyst on
subsequent development. J. Exp. Med. 140, 1049-1056.
Brook, F. A. and Gardner, R. L. (1997). The origin and efficient derivation of
embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709-5712.
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and
Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency
sustaining factor in embryonic stem cells. Cell 113, 643-655.
Chambers, I., Silva, J., Colby, D., Nichols, J., Nijmeijer, B., Robertson, M.,
Vrana, J., Jones, K., Grotewold, L. and Smith, A. (2007). Nanog safeguards
pluripotency and mediates germline development. Nature 450, 1230-1234.
Chang, H. H., Hemberg, M., Barahona, M., Ingber, D. E. and Huang, S.
(2008). Transcriptome-wide noise controls lineage choice in mammalian
progenitor cells. Nature 453, 544-547.
Chang, T. C., Yu, D., Lee, Y. S., Wentzel, E. A., Arking, D. E., West, K. M.,
Dang, C. V., Thomas-Tikhonenko, A. and Mendell, J. T. (2008). Widespread
microRNA repression by Myc contributes to tumorigenesis. Nat. Genet. 40, 43-
Chen, L., Glover, J. N., Hogan, P. G., Rao, A. and Harrison, S. C. (1998).
Structure of the DNA-binding domains from NFAT, Fos and Jun bound
specifically to DNA. Nature 392, 42-48.
Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V. B., Wong, E., Orlov, Y.
L., Zhang, W., Jiang, J. et al. (2008). Integration of external signaling pathways
with the core transcriptional network in embryonic stem cells. Cell 133, 1106-
Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J. J., Goldberg, R. B.,
Jacobsen, S. E. and Fischer, R. L. (2002). DEMETER, a DNA glycosylase domain
protein, is required for endosperm gene imprinting and seed viability in
arabidopsis. Cell 110, 33-42.
Chung, Y., Klimanskaya, I., Becker, S., Marh, J., Lu, S. J., Johnson, J.,
Meisner, L. and Lanza, R. (2006). Embryonic and extraembryonic stem cell
lines derived from single mouse blastomeres. Nature 439, 216-219.
Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M. and Zaret, K. S.
(2002). Opening of compacted chromatin by early developmental transcription
factors HNF3 (FoxA) and GATA-4. Mol. Cell 9, 279-289.
Conrad, S., Renninger, M., Hennenlotter, J., Wiesner, T., Just, L., Bonin, M.,
Aicher, W., Buhring, H. J., Mattheus, U., Mack, A. et al. (2008). Generation
of pluripotent stem cells from adult human testis. Nature 456, 344-349.
Cowan, C. A., Atienza, J., Melton, D. A. and Eggan, K. (2005). Nuclear
reprogramming of somatic cells after fusion with human embryonic stem cells.
Science 309, 1369-1373.
Davis, R. L., Weintraub, H. and Lassar, A. B. (1987). Expression of a single
transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000.
Deb-Rinker, P., Ly, D., Jezierski, A., Sikorska, M. and Walker, P. R. (2005).
Sequential DNA methylation of the Nanog and Oct-4 upstream regions in
human NT2 cells during neuronal differentiation. J. Biol. Chem. 280, 6257-6260.
Development 136 (4)
Development 136 (4)
Dietrich, J. E. and Hiiragi, T. (2007). Stochastic patterning in the mouse pre-
implantation embryo. Development 134, 4219-4231.
Dimos, J. T., Rodolfa, K. T., Niakan, K. K., Weisenthal, L. M., Mitsumoto, H.,
Chung, W., Croft, G. F., Saphier, G., Leibel, R., Goland, R. et al. (2008).
Induced pluripotent stem cells generated from patients with ALS can be
differentiated into motor neurons. Science 321, 1218-1221.
Do, J. T. and Scholer, H. R. (2004). Nuclei of embryonic stem cells reprogram
somatic cells. Stem Cells 22, 941-949.
Dominguez-Sola, D., Ying, C. Y., Grandori, C., Ruggiero, L., Chen, B., Li, M.,
Galloway, D. A., Gu, W., Gautier, J. and Dalla-Favera, R. (2007). Non-
transcriptional control of DNA replication by c-Myc. Nature 448, 445-451.
Dor, Y., Brown, J., Martinez, O. I. and Melton, D. A. (2004). Adult pancreatic
beta-cells are formed by self-duplication rather than stem-cell differentiation.
Nature 429, 41-46.
Durcova-Hills, G., Adams, I. R., Barton, S. C., Surani, M. A. and McLaren, A.
(2006). The role of exogenous fibroblast growth factor-2 on the reprogramming
of primordial germ cells into pluripotent stem cells. Stem Cells 24, 1441-1449.
Eggan, K., Baldwin, K., Tackett, M., Osborne, J., Gogos, J., Chess, A., Axel,
R. and Jaenisch, R. (2004). Mice cloned from olfactory sensory neurons. Nature
Egli, D., Rosains, J., Birkhoff, G. and Eggan, K. (2007). Developmental
reprogramming after chromosome transfer into mitotic mouse zygotes. Nature
Ellis, P., Fagan, B. M., Magness, S. T., Hutton, S., Taranova, O., Hayashi, S.,
McMahon, A., Rao, M. and Pevny, L. (2004). SOX2, a persistent marker for
multipotential neural stem cells derived from embryonic stem cells, the embryo
or the adult. Dev. Neurosci. 26, 148-165.
Eminli, S., Utikal, J., Arnold, K., Jaenisch, R. and Hochedlinger, K. (2008).
Reprogramming of neural progenitor cells into induced pluripotent stem cells in
the absence of exogenous Sox2 expression. Stem Cells 26, 2467-2474.
Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of
pluripotential cells from mouse embryos. Nature 292, 154-156.
Farthing, C. R., Ficz, G., Ng, R. K., Chan, C. F., Andrews, S., Dean, W.,
Hemberger, M. and Reik, W. (2008). Global mapping of DNA methylation in
mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS
Genet. 4, e1000116.
Feldman, N., Gerson, A., Fang, J., Li, E., Zhang, Y., Shinkai, Y., Cedar, H. and
Bergman, Y. (2006). G9a-mediated irreversible epigenetic inactivation of Oct-
3/4 during early embryogenesis. Nat. Cell Biol. 8, 188-194.
Feng, R., Desbordes, S. C., Xie, H., Tillo, E. S., Pixley, F., Stanley, E. R. and
Graf, T. (2008). PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-
like cells. Proc. Natl. Acad. Sci. USA 105, 6057-6062.
Finch, B. W. and Ephrussi, B. (1967). Retention of multiple developmental
potentialities by cells of a mouse testicular teratocarcinoma during prolonged
culture in vitro and their extinction upon hybridization with cells of permanent
lines. Proc. Natl. Acad. Sci. USA 57, 615-621.
Fuhrmann, G., Chung, A. C., Jackson, K. J., Hummelke, G., Baniahmad, A.,
Sutter, J., Sylvester, I., Scholer, H. R. and Cooney, A. J. (2001). Mouse
germline restriction of Oct4 expression by germ cell nuclear factor. Dev. Cell 1,
Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K.,
Miyazaki Ji, J. and Niwa, H. (2002). Differentiation of embryonic stem cells is
induced by GATA factors. Genes Dev. 16, 784-789.
Galan-Caridad, J. M., Harel, S., Arenzana, T. L., Hou, Z. E., Doetsch, F. K.,
Mirny, L. A. and Reizis, B. (2007). Zfx controls the self-renewal of embryonic
and hematopoietic stem cells. Cell 129, 345-357.
Gong, Z., Morales-Ruiz, T., Ariza, R. R., Roldan-Arjona, T., David, L. and Zhu,
J. K. (2002). ROS1, a repressor of transcriptional gene silencing in Arabidopsis,
encodes a DNA glycosylase/lyase. Cell 111, 803-814.
Guan, K., Nayernia, K., Maier, L. S., Wagner, S., Dressel, R., Lee, J. H., Nolte,
J., Wolf, F., Li, M., Engel, W. et al. (2006). Pluripotency of spermatogonial
stem cells from adult mouse testis. Nature 440, 1199-1203.
Gurdon, J. (1962). The developmental capacity of nuclei taken from intestinal
epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 10, 622-640.
Hajkova, P., Ancelin, K., Waldmann, T., Lacoste, N., Lange, U. C., Cesari, F.,
Lee, C., Almouzni, G., Schneider, R. and Surani, M. A. (2008). Chromatin
dynamics during epigenetic reprogramming in the mouse germ line. Nature 452,
Hanna, J., Wernig, M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J.
P., Beard, C., Brambrink, T., Wu, L. C., Townes, T. M. et al. (2007). Treatment
of sickle cell anemia mouse model with iPS cells generated from autologous
skin. Science 318, 1920-1923.
Hanna, J., Markoulaki, S., Schorderet, P., Carey, B. W., Beard, C., Wernig, M.,
Creyghton, M. P., Steine, E. J., Cassady, J. P., Foreman, R. et al. (2008).
Direct reprogramming of terminally differentiated mature B lymphocytes to
pluripotency. Cell 133, 250-264.
Hayashi, K., Lopes, S. M., Tang, F. and Surani, M. A. (2008). Dynamic
equilibrium and heterogeneity of mouse pluripotent stem cells with distinct
functional and epigenetic states. Cell Stem Cell 3, 391-401.
Hendrich, B., Guy, J., Ramsahoye, B., Wilson, V. A. and Bird, A. (2001). Closely
related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse
development. Genes Dev. 15, 710-723.
Henikoff, S. and Ahmad, K. (2005). Assembly of variant histones into chromatin.
Annu. Rev. Cell Dev. Biol. 21, 133-153.
Hochedlinger, K. and Jaenisch, R. (2002a). Monoclonal mice generated by
nuclear transfer from mature B and T donor cells. Nature 415, 1035-1038.
Hochedlinger, K. and Jaenisch, R. (2002b). Nuclear transplantation: lessons from
frogs and mice. Curr. Opin. Cell Biol. 14, 741-748.
Hochedlinger, K. and Jaenisch, R. (2006). Nuclear reprogramming and
pluripotency. Nature 441, 1061-1067.
Hochedlinger, K. and Jaenisch, R. (2007). On the cloning of animals from
terminally differentiated cells. Nat. Genet. 39, 136-137; author reply 137-138.
Hochedlinger, K., Blelloch, R., Brennan, C., Yamada, Y., Kim, M., Chin, L. and
Jaenisch, R. (2004). Reprogramming of a melanoma genome by nuclear
transplantation. Genes Dev. 18, 1875-1885.
Hockemeyer, D., Soldner, F., Cook, E. G., Gao, Q., Mitalipova, M. and
Jaenisch, R. (2008). A drug-inducible system for direct reprogramming of
human somatic cells to pluripotency. Cell Stem Cell 3, 346-353.
Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E.
and Melton, D. A. (2008a). Induction of pluripotent stem cells by defined
factors is greatly improved by small-molecule compounds. Nat. Biotechnol. 26,
Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S.,
Muhlestein, W. and Melton, D. A. (2008b). Induction of pluripotent stem cells
from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26,
Imamura, M., Miura, K., Iwabuchi, K., Ichisaka, T., Nakagawa, M., Lee, J.,
Kanatsu-Shinohara, M., Shinohara, T. and Yamanaka, S. (2006).
Transcriptional repression and DNA hypermethylation of a small set of ES cell
marker genes in male germline stem cells. BMC Dev. Biol. 6, 34.
Inoue, K., Wakao, H., Ogonuki, N., Miki, H., Seino, K., Nambu-Wakao, R.,
Noda, S., Miyoshi, H., Koseki, H., Taniguchi, M. et al. (2005). Generation of
cloned mice by direct nuclear transfer from natural killer T cells. Curr. Biol. 15,
Inoue, K., Ogonuki, N., Miki, H., Hirose, M., Noda, S., Kim, J. M., Aoki, F.,
Miyoshi, H. and Ogura, A. (2006). Inefficient reprogramming of the
hematopoietic stem cell genome following nuclear transfer. J. Cell Sci. 119,
Jaenisch, R. and Bird, A. (2003). Epigenetic regulation of gene expression: how
the genome integrates intrinsic and environmental signals. Nat. Genet. 33
Jaenisch, R. and Young, R. (2008). Stem cells, the molecular circuitry of
pluripotency and nuclear reprogramming. Cell 132, 567-582.
Jin, S. G., Guo, C. and Pfeifer, G. P. (2008). GADD45A does not promote DNA
demethylation. PLoS Genet. 4, e1000013.
Kahan, B. W. and Ephrussi, B. (1970). Developmental potentialities of clonal in
vitro cultures of mouse testicular teratoma. J. Natl. Cancer Inst. 44, 1015-1036.
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki,
H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S. et al. (2004). Generation of
pluripotent stem cells from neonatal mouse testis. Cell 119, 1001-1012.
Kangaspeska, S., Stride, B., Metivier, R., Polycarpou-Schwarz, M., Ibberson,
D., Carmouche, R. P., Benes, V., Gannon, F. and Reid, G. (2008). Transient
cyclical methylation of promoter DNA. Nature 452, 112-115.
Kim, J., Chu, J., Shen, X., Wang, J. and Orkin, S. H. (2008a). An extended
transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049-
Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-
Bravo, M. J., Ruau, D., Han, D. W., Zenke, M. et al. (2008b). Pluripotent stem
cells induced from adult neural stem cells by reprogramming with two factors.
Nature 454, 646-650.
Kleinsmith, L. J. and Pierce, G. B., Jr (1964). Multipotentiality of Single
Embryonal Carcinoma Cells. Cancer Res. 24, 1544-1551.
Knoepfler, P. S. (2008). Why myc? An unexpected ingredient in the stem cell
cocktail. Cell Stem Cell 2, 18-21.
Kopp, J. L., Ormsbee, B. D., Desler, M. and Rizzino, A. (2008). Small increases
in the level of sox2 trigger the differentiation of mouse embryonic stem cells.
Stem Cells 26, 903-911.
Kuramochi-Miyagawa, S., Watanabe, T., Gotoh, K., Totoki, Y., Toyoda, A.,
Ikawa, M., Asada, N., Kojima, K., Yamaguchi, Y., Ijiri, T. W. et al. (2008).
DNA methylation of retrotransposon genes is regulated by Piwi family members
MILI and MIWI2 in murine fetal testes. Genes Dev. 22, 908-917.
Kustikova, O., Fehse, B., Modlich, U., Yang, M., Dullmann, J., Kamino, K.,
von Neuhoff, N., Schlegelberger, B., Li, Z. and Baum, C. (2005). Clonal
dominance of hematopoietic stem cells triggered by retroviral gene marking.
Science 308, 1171-1174.
Laiosa, C. V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L. and Graf, T. (2006).
Reprogramming of committed T cell progenitors to macrophages and dendritic
cells by C/EBP alpha and PU.1 transcription factors. Immunity 25, 731-744.
Lam, M. Y. and Nadeau, J. H. (2003). Genetic control of susceptibility to
spontaneous testicular germ cell tumors in mice. Apmis 111, 184-190;
Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R.
M., Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K. et al. (2006).
Control of developmental regulators by Polycomb in human embryonic stem
cells. Cell 125, 301-313.
Lehnertz, B., Ueda, Y., Derijck, A. A., Braunschweig, U., Perez-Burgos, L.,
Kubicek, S., Chen, T., Li, E., Jenuwein, T. and Peters, A. H. (2003). Suv39h-
mediated histone H3 lysine 9 methylation directs DNA methylation to major
satellite repeats at pericentric heterochromatin. Curr. Biol. 13, 1192-1200.
Li, J., Ishii, T., Feinstein, P. and Mombaerts, P. (2004). Odorant receptor gene
choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature
Li, J., Greco, V., Guasch, G., Fuchs, E. and Mombaerts, P. (2007). Mice cloned
from skin cells. Proc. Natl. Acad. Sci. USA 104, 2738-2743.
Li, L., Connelly, M. C., Wetmore, C., Curran, T. and Morgan, J. I. (2003).
Mouse embryos cloned from brain tumors. Cancer Res. 63, 2733-2736.
Liang, J., Wan, M., Zhang, Y., Gu, P., Xin, H., Jung, S. Y., Qin, J., Wong, J.,
Cooney, A. J., Liu, D. et al. (2008). Nanog and Oct4 associate with unique
transcriptional repression complexes in embryonic stem cells. Nat. Cell Biol. 10,
Lin, I. G., Tomzynski, T. J., Ou, Q. and Hsieh, C. L. (2000). Modulation of DNA
binding protein affinity directly affects target site demethylation. Mol. Cell. Biol.
Lin, T., Chao, C., Saito, S., Mazur, S. J., Murphy, M. E., Appella, E. and Xu, Y.
(2005). p53 induces differentiation of mouse embryonic stem cells by
suppressing Nanog expression. Nat. Cell Biol. 7, 165-171.
Lluis, F., Pedone, E., Pepe, S. and Cosma, M. P. (2008). Periodic activation of
Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by
cell fusion. Cell Stem Cell 3, 493-507.
Loh, Y. H., Wu, Q., Chew, J. L., Vega, V. B., Zhang, W., Chen, X., Bourque, G.,
George, J., Leong, B., Liu, J. et al. (2006). The Oct4 and Nanog transcription
network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38,
Lowry, W. E., Richter, L., Yachechko, R., Pyle, A. D., Tchieu, J., Sridharan, R.,
Clark, A. T. and Plath, K. (2008). Generation of human induced pluripotent
stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105, 2883-2888.
Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K.,
Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R. et al. (2007). Directly
reprogrammed fibroblasts show global epigenetic reprogramming and
widespread tissue contribution. Cell Stem Cell 1, 55-70.
Maherali, N., Ahfeldt, T., Rigamonti, A., Utikal, J., Cowan, C. and
Hochedlinger, K. (2008). A high-efficiency system for the generation and study
of human induced pluripotent stem cells. Cell Stem Cell 3, 340-345.
Marson, A., Foreman, R., Chevalier, B., Bilodeau, S., Kahn, M., Young, R. A.
and Jaenisch, R. (2008). Wnt signaling promotes reprogramming of somatic
cells to pluripotency. Cell Stem Cell 3, 132-135.
Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad.
Sci. USA 78, 7634-7638.
Matsui, Y., Zsebo, K. and Hogan, B. L. (1992). Derivation of pluripotential
embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841-
Meissner, A., Wernig, M. and Jaenisch, R. (2007). Direct reprogramming of
genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol.
Merika, M., Williams, A. J., Chen, G., Collins, T. and Thanos, D. (1998).
Recruitment of CBP/p300 by the IFN beta enhanceosome is required for
synergistic activation of transcription. Mol. Cell 1, 277-287.
Metivier, R., Gallais, R., Tiffoche, C., Le Peron, C., Jurkowska, R. Z.,
Carmouche, R. P., Ibberson, D., Barath, P., Demay, F., Reid, G. et al. (2008).
Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45-
Mikkelsen, T. S., Hanna, J., Zhang, X., Ku, M., Wernig, M., Schorderet, P.,
Bernstein, B. E., Jaenisch, R., Lander, E. S. and Meissner, A. (2008).
Dissecting direct reprogramming through integrative genomic analysis. Nature
Miller, R. A. and Ruddle, F. H. (1976). Pluripotent teratocarcinoma-thymus
somatic cell hybrids. Cell 9, 45-55.
Mintz, B. and Illmensee, K. (1975). Normal genetically mosaic mice produced
from malignant teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 72, 3585-3589.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K.,
Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein
Nanog is required for maintenance of pluripotency in mouse epiblast and ES
cells. Cell 113, 631-642.
Mohn, F., Weber, M., Rebhan, M., Roloff, T. C., Richter, J., Stadler, M. B.,
Bibel, M. and Schubeler, D. (2008). Lineage-specific polycomb targets and de
novo DNA methylation define restriction and potential of neuronal progenitors.
Mol. Cell 30, 755-766.
Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T.,
Okita, K., Mochiduki, Y., Takizawa, N. and Yamanaka, S. (2008). Generation
of induced pluripotent stem cells without Myc from mouse and human
fibroblasts. Nat. Biotechnol. 26, 101-106.
Ng, R. K. and Gurdon, J. B. (2005). Epigenetic memory of active gene
transcription is inherited through somatic cell nuclear transfer. Proc. Natl. Acad.
Sci. USA 102, 1957-1962.
Ng, R. K. and Gurdon, J. B. (2008). Epigenetic memory of an active gene state
depends on histone H3.3 incorporation into chromatin in the absence of
transcription. Nat. Cell Biol. 10, 102-109.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D.,
Chambers, I., Scholer, H. and Smith, A. (1998). Formation of pluripotent stem
cells in the mammalian embryo depends on the POU transcription factor Oct4.
Cell 95, 379-391.
Niwa, H., Miyazaki, J. and Smith, A. G. (2000). Quantitative expression of Oct-
3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat.
Genet. 24, 372-376.
Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R.
and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines
trophectoderm differentiation. Cell 123, 917-929.
Nutt, S. L., Heavey, B., Rolink, A. G. and Busslinger, M. (1999). Commitment
to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401,
Ohinata, Y., Payer, B., O’Carroll, D., Ancelin, K., Ono, Y., Sano, M., Barton, S.
C., Obukhanych, T., Nussenzweig, M., Tarakhovsky, A. et al. (2005). Blimp1
is a critical determinant of the germ cell lineage in mice. Nature 436, 207-213.
Okita, K., Ichisaka, T. and Yamanaka, S. (2007). Generation of germline-
competent induced pluripotent stem cells. Nature 448, 313-317.
Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. and Yamanaka, S.
(2008). Generation of mouse induced pluripotent stem cells without viral
vectors. Science 322, 949-953.
Orkin, S. H. and Zon, L. I. (2008). Hematopoiesis: an evolving paradigm for stem
cell biology. Cell 132, 631-644.
Panne, D., Maniatis, T. and Harrison, S. C. (2004). Crystal structure of ATF-2/c-
Jun and IRF-3 bound to the interferon-beta enhancer. EMBO J. 23, 4384-4393.
Panne, D., Maniatis, T. and Harrison, S. C. (2007). An atomic model of the
interferon-beta enhanceosome. Cell 129, 1111-1123.
Park, I. H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A.,
Lensch, M. W., Cowan, C., Hochedlinger, K. and Daley, G. Q. (2008a).
Disease-specific induced pluripotent stem cells. Cell 134, 877-886.
Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P.
H., Lensch, M. W. and Daley, G. Q. (2008b). Reprogramming of human
somatic cells to pluripotency with defined factors. Nature 451, 141-146.
Puri, P. L., Iezzi, S., Stiegler, P., Chen, T. T., Schiltz, R. L., Muscat, G. E.,
Giordano, A., Kedes, L., Wang, J. Y. and Sartorelli, V. (2001). Class I histone
deacetylases sequentially interact with MyoD and pRb during skeletal
myogenesis. Mol. Cell 8, 885-897.
Resnick, J. L., Bixler, L. S., Cheng, L. and Donovan, P. J. (1992). Long-term
proliferation of mouse primordial germ cells in culture. Nature 359, 550-551.
Santos, F., Hendrich, B., Reik, W. and Dean, W. (2002). Dynamic
reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241,
Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A. H. (2004).
Maintenance of pluripotency in human and mouse embryonic stem cells
through activation of Wnt signaling by a pharmacological GSK-3-specific
inhibitor. Nat. Med. 10, 55-63.
Seandel, M., James, D., Shmelkov, S. V., Falciatori, I., Kim, J., Chavala, S.,
Scherr, D. S., Zhang, F., Torres, R., Gale, N. W. et al. (2007). Generation of
functional multipotent adult stem cells from GPR125+ germline progenitors.
Nature 449, 346-350.
Sharif, J., Muto, M., Takebayashi, S., Suetake, I., Iwamatsu, A., Endo, T. A.,
Shinga, J., Mizutani-Koseki, Y., Toyoda, T., Okamura, K. et al. (2007). The
SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to
methylated DNA. Nature 450, 908-912.
Shi, Y., Desponts, C., Do, J. T., Hahm, H. S., Scholer, H. R. and Ding, S.
(2008a). Induction of pluripotent stem cells from mouse embryonic fibroblasts
by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568-574.
Shi, Y., Do, J. T., Desponts, C., Hahm, H. S., Scholer, H. R. and Ding, S.
(2008b). A combined chemical and genetic approach for the generation of
induced pluripotent stem cells. Cell Stem Cell 2, 525-528.
Shimosato, D., Shiki, M. and Niwa, H. (2007). Extra-embryonic endoderm cells
derived from ES cells induced by GATA factors acquire the character of XEN cells.
BMC Dev. Biol. 7, 80.
Silva, J., Chambers, I., Pollard, S. and Smith, A. (2006). Nanog promotes
transfer of pluripotency after cell fusion. Nature 441, 997-1001.
Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T. W. and
Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by
signal inhibition. PLoS Biol. 6, e253.
Simonsson, S. and Gurdon, J. (2004). DNA demethylation is necessary for the
epigenetic reprogramming of somatic cell nuclei. Nat. Cell. Biol. 6, 984-990.
Development 136 (4)
Development 136 (4)
Stadtfeld, M., Brennand, K. and Hochedlinger, K. (2008a). Reprogramming of
pancreatic beta cells into induced pluripotent stem cells. Curr. Biol. 18, 890-894.
Stadtfeld, M., Maherali, N., Breault, D. T. and Hochedlinger, K. (2008b).
Defining molecular cornerstones during fibroblast to iPS cell reprogramming in
mouse. Cell Stem Cell 2, 230-240.
Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. and Hochedlinger, K. (2008c).
Induced pluripotent stem cells generated without viral integration. Science 322,
Stevens, L. C. (1967). Origin of testicular teratomas from primordial germ cells in
mice. J. Natl. Cancer Inst. 38, 549-552.
Stevens, L. C. and Little, C. C. (1954). Spontaneous testicular teratomas in an
inbred strain of mice. Proc. Natl. Acad. Sci. USA 40, 1080-1087.
Sung, L. Y., Gao, S., Shen, H., Yu, H., Song, Y., Smith, S. L., Chang, C. C.,
Inoue, K., Kuo, L., Lian, J. et al. (2006). Differentiated cells are more efficient
than adult stem cells for cloning by somatic cell nuclear transfer. Nat. Genet. 38,
Surani, M. A., Hayashi, K. and Hajkova, P. (2007). Genetic and epigenetic
regulators of pluripotency. Cell 128, 747-762.
Tada, M., Tada, T., Lefebvre, L., Barton, S. C. and Surani, M. A. (1997).
Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in
hybrid cells. EMBO J. 16, 6510-6520.
Tada, M., Takahama, Y., Abe, K., Nakatsuji, N. and Tada, T. (2001). Nuclear
reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol.
Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-
Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K.
and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell 131, 861-872.
Thomassin, H., Flavin, M., Espinas, M. L. and Grange, T. (2001).
Glucocorticoid-induced DNA demethylation and gene memory during
development. EMBO J. 20, 1974-1983.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel,
J. J., Marshall, V. S. and Jones, J. M. (1998). Embryonic stem cell lines derived
from human blastocysts. Science 282, 1145-1147.
Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K. and Niwa, H.
(2008). Identification and characterization of subpopulations in undifferentiated
ES cell culture. Development 135, 909-918.
Varas, F., Stadtfeld, M., De Andres-Aguayo, L., Maherali, N., di, Tullio, A.,
Pantano, L., Notredame, C., Hochedlinger, K. and Graf, T. (2008). Fibroblast
derived induced pluripotent stem cells show no common retroviral vector
insertions. Stem Cells doi: 10.1634/stemcells.2008-0696.
Viswanathan, S. R., Daley, G. Q. and Gregory, R. I. (2008). Selective blockade
of microRNA processing by Lin28. Science 320, 97-100.
Waddington, C. H. (1957). The Strategy of the Genes. London: Geo Allen and
Wakayama, S., Hikichi, T., Suetsugu, R., Sakaide, Y., Bui, H. T., Mizutani, E.
and Wakayama, T. (2007). Efficient establishment of mouse embryonic stem
cell lines from single blastomeres and polar bodies. Stem Cells 25, 986-993.
Wakayama, T. and Yanagimachi, R. (1999). Cloning of male mice from adult
tail-tip cells. Nat. Genet. 22, 127-128.
Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D. N., Theunissen, T. W. and
Orkin, S. H. (2006). A protein interaction network for pluripotency of
embryonic stem cells. Nature 444, 364-368.
Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M.,
Hochedlinger, K., Bernstein, B. E. and Jaenisch, R. (2007). In vitro
reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448,
Wernig, M., Lengner, C. J., Hanna, J., Lodato, M. A., Steine, E. J., Foreman,
R., Staerk, J., Markoulaki, S. and Jaenisch, R. (2008a). A drug-inducible
transgenic system for direct reprogramming of multiple somatic cell types. Nat.
Biotechnol. 26, 916-924.
Wernig, M., Meissner, A., Cassady, J. P. and Jaenisch, R. (2008b). c-Myc is
dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10-
Wernig, M., Zhao, J. P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli,
V., Constantine-Paton, M., Isacson, O. and Jaenisch, R. (2008c). Neurons
derived from reprogrammed fibroblasts functionally integrate into the fetal brain
and improve symptoms of rats with Parkinson’s disease. Proc. Natl. Acad. Sci.
USA 105, 5856-5861.
Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. and Campbell, K. H. (1997).
Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810-
Xie, H., Ye, M., Feng, R. and Graf, T. (2004). Stepwise reprogramming of B cells
into macrophages. Cell 117, 663-676.
Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J.,
Cohen, P. and Smith, A. (2008). The ground state of embryonic stem cell self-
renewal. Nature 453, 519-523.
Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.
L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R. et al. (2007).
Induced pluripotent stem cell lines derived from human somatic cells. Science
Zhao, Y., Yin, X., Qin, H., Zhu, F., Liu, H., Yang, W., Zhang, Q., Xiang, C., Hou,
P., Song, Z. et al. (2008). Two supporting factors greatly improve the efficiency
of human iPSC generation. Cell Stem Cell 3, 475-479.
Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. and Melton, D. A. (2008). In
vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455,
Zhu, H., Geiman, T. M., Xi, S., Jiang, Q., Schmidtmann, A., Chen, T., Li, E. and
Muegge, K. (2006). Lsh is involved in de novo methylation of DNA. EMBO J.