NEWS & VIEWS

ST E M CELLS

A designers guide to pluripotency

Pluripotent stem cells, which give rise to almost all cell types, can be engineered from mature cells. A thorough analysis of
the process has led to the characterization of a new type of pluripotent cell. See Articles p.192 & p.198
JUN WU & JUAN CARLOS IZPISUA BELMONTE

luripotency, defined as the ability of

a cell to generate all cell types in the
adult organism, is a transient feature of
early embryonic development. Two distinct
pluripotent cell types can be isolated from
embryos and cultured in vitro14 naive
cells, called embryonic stem cells, and those
primed for differentiation, epiblast stem cells.
Furthermore, a defined cocktail of transcription factors, called reprogramming factors, can
reinstate pluripotency when introduced into
mature cells, producing induced pluripotent
stem cells (iPSCs)57. In addition to known
pluripotent cell types5,8, iPSC generation yields
a spectrum of distinct cell types, hinting at
the existence of uncharacterized pluripotent
states. A collection of five manuscripts (two
in this issue9,10 and three in Nature Communications1113), now uncover and characterize
an alternative pluripotent outcome of iPSC
reprogramming: F-class cells (Fig. 1).
These five manuscripts are part of an international collaboration called Project Grandiose, in which the researchers set out to
reanalyse the process of iPSC reprogramming
from an unbiased perspective. They reasoned
that, by extensively documenting the molecular and cellular transitions occurring at each
stage of the process, they could provide both
the first thorough roadmap for iPSC reprogramming, and an explanation for the emergence during reprogramming of undefined
pluripotent cell types, which have been mostly
overlooked by previous studies.
In the first paper, Tonge et al.9 (page 192)
identify F-class cells named because of
their unusual, fuzzy-looking colony morphology as a pluripotent cell type distinct
from embryonic stem cells (ES cells) and
epiblast stem cells. Maintenance of F-class
cells depends on continuing high expression
of reprogramming factors. In conventional
reprogramming methods, the expression of
introduced genes (transgenes) is silenced by
factors that are expressed in the host cells
once pluripotency is achieved, and thus
F-class cells could not have been identified in
those assays. The researchers use of a hostfactor-independent reprogramming method
bypasses transgene silencing and thereby

E3.5

Embryo

E5.5

Stem-cell
culture

ES cells

Low levels of
reprogramming
factors

EpiSCs

F-class cells
High levels of
reprogramming
factors
Mature-cell
culture

Figure 1 | Different flavours of pluripotency. Two distinct types of pluripotent stem cell have
been captured from early mouse embryos for culture in vitro embryonic stem cells (ES cells) from
embryos at three-and-a-half days old (E3.5), and epiblast stem cells (EpiSCs) from embryos at E5.5.
The pluripotent-cell populations in each embryo are shown in blue. These two cell types can also be
induced from mature cells through cellular reprogramming using low levels of reprogramming factors.
Five papers913 from Project Grandiose investigate the molecular details of cellular reprogramming, and
uncover a new type of pluripotent cell, dubbed F-class, which depends on sustained, high-level expression
of reprogramming factors. This discovery hints at the potential that other, unidentified pluripotent states
exist (marked with a question mark), and might either be generated by engineering or be present in the
early embryo.

allows sustained high-level expression of

reprogramming factors14.
Tonge and colleagues report that the fuzzy
morphology of F-class cells arises from their
low adhesiveness, which, along with their fast
proliferation, makes these cells more amenable to large-scale production than EScells.
This is a desirable feature for cell-based
therapies, which demand large quantities of
specific cell types. For example, pancreatic
-cells, which store and release insulin, can be
derived from pluripotent cells and might be
used to treat people with diabetes15. However,
F-class cells dependence on transgenes could
be problematic for their safe clinical application, because mutations arising from either
improper transgene insertion into the genome

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or incomplete inactivation of reprogramming

factors when the cells begin differentiation
might ultimately lead to tumour formation.
One solution might be to stabilize the F-state
independent of transgenes, using small molecules. This strategy has been successful for
stabilizing naive-like human pluripotent stem
cells16,17. Tonge et al. show that ES-like cells
convert to the F-state following forced expression of reprogramming factors. Conversely,
F-class cells can be converted to an ES-celllike state using small molecules that inhibit
the activity of a class of enzymes called histone
deacetylases, which modulate gene expression
by removing acetyl molecules from the histone
proteins around which DNA is packaged.
Such interconvertibility may lead to insights

NEWS & VIEWS RESEARCH

into how pluripotency is stabilized in distinct
cellular contexts.
In the second paper, Hussein et al. 10
(page 198) define the different molecular routes
to pluripotency by performing the most detailed
analysis of reprogramming so far. Among other
findings, the authors uncover key determinants
for the emergence of ES-cell-like or F-class
states. Emergence of the F-class state relies on
repression of genes that are expressed in ES
cells. This is achieved through a molecular
modification associated with gene repression
the attachment of three methyl molecules
to an amino-acid residue, lysine 27, of histone
H3 proteins. By contrast, the loss of the DNA
methylation marks inherited from mature cells
is necessary for cells to take on an ES-cell-like
state, but some of these marks are retained in
F-class cells.
The remaining three studies complement
Hussein and colleagues work by providing
descriptive, in-depth analyses of the changes
in molecular pathways en route to pluripotency, generating large data sets that are freely
available at www.stemformatics.org. Lee et al.11
interrogate the epigenetic changes (those
modifications to the genome that affect gene
expression without altering DNA sequence)
that occur during the transition to pluripotency. They conclude that DNA methylation
has a crucial role in iPSC reprogramming
and acts as an epigenetic switch between
F-class and ES-cell-like states. Clancy and
colleagues12 delineate the dynamic changes
in small RNAs post-transcriptional regulators of gene expression during iPSC reprogramming, and find that a distinct group of

microRNAs supports the F-class pluripotency

program. Finally, Benevento et al.13 show that
reorganization of protein expression occurs
in two defined waves during cellular reprogramming. The authors show that patterns of
protein expression differ between ES-cell-like
and F-class states.
These five manuscripts mark the first steps
towards understanding F-class pluripotency
and thus towards making the most of their
clinical potential. The molecular mechanisms
underpinning the F-state warrant further
investigation, as do the metabolic cues that
contribute to sustaining F-class cells, because
different pluripotent stem cells probably have
distinct metabolic requirements18. Remaining
questions include whether human F-class cells
can be generated through cellular reprogramming, and if functional differentiated cells can
be obtained from F-class cells.
In embracing the inherent artificiality of
iPSC reprogramming, Project Grandiose
has opened up the field to fresh avenues of
research. This work shows that a third pluripotent state can be engineered in vitro, and it may
be that there are other pluripotent endpoints of
reprogramming (Fig. 1). Moreover, there may
be other pluripotent states in the developing
embryo. If there are, it would be interesting to
determine whether such states could be captured and cultured in vitro. To investigate these
avenues, an unbiased approach, such as that
taken by Tonge et al., will probably prevail.
Looking ahead, customized stem cells
designed for specific applications such as
large-scale expansion, or fast, synchronized
differentiation may soon become a reality.

MAT ERIALS SCIENCE

Breakthrough
for protons
The atomically thin material called graphene is impermeable to atoms as small as
helium. The finding that protons can pass through it might enable new kinds of
membrane to be developed and aid research into fuel cells. See Letter p.227
ROHIT N. KARNIK

he two-dimensional material graphene

is often depicted as a hexagonal mesh
of carbon atoms, with plenty of space
between its atoms. But in reality, the finite
size of the carbon atoms leaves little room for
anything to slip through. In 2008, a classic
experiment1 revealed that pristine graphene
is impermeable to helium and other gases at
room temperature, making it the thinnest
barrier known to science. The results logically
extend to other two-dimensional materials,

including hexagonal boron nitride (hBN) and

molybdenum disulphide (MoS2). By contrast,
in a paper published in this issue (page 227),
Hu et al.2 present the unexpected finding that
graphene and hBN but not MoS2 are
excellent conductors of protons across their
two-dimensional structure.
The authors measured the electric current
across micrometre-sized flakes of graphene,
hBN or MoS2 sandwiched between two layers
of a polymer that conducts protons when
hydrated (that is, in the presence of water).
In the absence of other charge carriers, the

The existence of alternative pluripotent states

adds another dimension to the potential of
pluripotent stem cells in regenerative medicine. The results of Project Grandiose call for
future work that catalogues myriad molecularly and functionally distinct pluripotent stem
cells to harness their full potential.
Jun Wu and Juan Carlos Izpisua Belmonte
are in the Gene Expression Laboratory, Salk
Institute for Biological Studies, La Jolla,
California 92037, USA.
e-mail: belmonte@salk.edu
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measured current is a direct indicator of proton transport. Hu and colleagues detected

substantial current across graphene, and an
even higher current across hBN, but no current across MoS2 indicating that graphene
and hBN conduct protons, but MoS2 does not.
Any contribution to the proton conductivity from defects in the two-dimensional
materials can be ruled out, because the results
were remarkably repeatable across different experiments and because the researchers
carefully characterized the materials. The same
conductivities were obtained when aqueous
hydrochloric acid a source of protons was
placed on either side of the materials, showing that the proton conductivity was a general
effect and was not limited to the experiment
with polymer layers. Hu and co-workers went
on to show that bilayers and trilayers of hBN
conduct protons, albeit with reduced conductivity compared with monolayers. However,
in the case of graphene, even one extra layer
entirely obliterates proton conductivity, so that
bilayer graphene is essentially impermeable.
The observed proton conductivities
or lack thereof can be explained by the
electron-density distribution in the

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