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: firstname.lastname@example.org; email@example.com
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
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2006 First induced pluripotent stem (iPS) cells generated from adult
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