X-chromosome epigenetic reprogramming in pluripotent stem cells via noncoding genes.

Abstract

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally 'naïve', with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.

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Review
X-chromosome epigenetic reprogramming in pluripotent stem cells via
noncoding genes
Daniel H. Kima, Yesu Jeonb, Montserrat C. Anguerab, Jeannie T. Leeb,∗
aDivision of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA
bHoward Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, 185 Cambridge St.,
Boston, MA 02114, USA
a r t i c l e i n f o
Article history:
Available online xxx
Keywords:
Epigenetics
Reprogramming
X-chromosome inactivation
Pluripotency
Stem cells
Noncoding RNAs
a b s t r a c t
Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome.
Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell dif-
ferentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of
the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem
cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation
center. This review article will discuss the latest advances in our understanding at the molecular level.
Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is char-
acterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the
X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability
that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks
on the X-chromosome indicate whether human pluripotent stem cells are developmentally ‘naïve’, with
characteristicsofthepluripotentgroundstate.X-chromosomestatusanddeterminationthereofvianon-
coding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem
cells, an important consideration given their enormous potential for stem cell therapy.
© 2011 Published by Elsevier Ltd.
Contents
1.
2.
Introduction..........................................................................................................................................
Mouse X-chromosome regulation ...................................................................................................................
2.1. Mouse ES cells ................................................................................................................................
2.2. Mouse iPS cells ...............................................................................................................................
2.3.Mouse EpiSCs.................................................................................................................................
Human X-chromosome regulation ..................................................................................................................
3.1. Human ES cells ...............................................................................................................................
3.2.Naïve human ES cells.........................................................................................................................
3.3. Human iPS cells...............................................................................................................................
Conclusions ..........................................................................................................................................
Acknowledgements..................................................................................................................................
References ...........................................................................................................................................
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3.
4.
1. Introduction
This review article will discuss the tight linkage between X-
chromosome and stem cell reprogramming. Recent studies have
shown that this linkage is mediated by pluripotency factors act-
∗Corresponding author. Tel.: +1 617 726 5943; fax: +1 617 726 6893.
E-mail addresses: dkim@caltech.edu (D.H. Kim), lee@molbio.mgh.harvard.edu
(J.T. Lee).
ing specifically on noncoding genes of the X-inactivation center
(Xic) to initiate or reverse X-chromosome inactivation (XCI), the
mechanism of dosage compensation in mammals which leads to
transcriptional inactivation of one X-chromosome in the female.
XCI provides a classic model for noncoding RNA (ncRNA)-mediated
epigenetic regulation [1–3]. These ncRNAs are located at the Xic,
a regulatory hub that mediates the stepwise formation of Xi hete-
rochromatin [4]. The onset of XCI corresponds with expression of
the17-kbnoncodingXistRNA,whichcoatstheentireinactiveX(Xi)
chromosome in cis [5–11]. Xist mediates facultative heterochro-
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matinontheXithroughrecruitmentandinteractionwithPolycomb
group proteins [12], marking the Xi with histone H3 lysine 27
trimethylation (H3K27me3) [13–15]. Xist expression is regulated
by three other ncRNAs, with two functioning in the activation of
Xist (RepA, Jpx) [12,16,17] and one functioning to antagonize its
activation (Tsix) [18–20].
Although thisreviewwill
chromosome inactivation, it should be briefly mentioned that
XCI can be subject to parental imprinting in marsupial mam-
mals and also in the extraembryonic lineages of some eutherian
mammals (e.g., mouse, cow) [21,22]. Imprinted XCI occurs on
the paternal X-chromosome and is believed to be the ancestral
form of mammalian dosage compensation. In mice, the imprinted
form of XCI is observed first during development in all cells,
but persists only in the extraembryonic tissues after embry-
onic day 4.5, when imprint erasure and X-reactivation occur in
the epiblast lineage [23–26]. Among ncRNAs involved in “ran-
dom” XCI, Xist and Tsix are thus far the only ones known to
also participate in imprinted XCI. Embryos lacking Tsix cannot
protect the maternal X-chromosome from silencing [20,27], and
those lacking Xist cannot initiate genic silencing on the paternal
X [10,25].
Following reactivation of the paternal X-chromosome, cells of
the epiblast lineage undergo random XCI and give rise to the
embryo proper. From mouse and human embryos, it is possible
to derive cells from this lineage and generate embryonic stem (ES)
cells, a pluripotent cell type capable of differentiating into all three
germ lineages (ectoderm, mesoderm, endoderm). ES cells have
providedavaluableexvivosystemforthestudyofepigeneticrepro-
grammingandtheroleofXCIandncRNAsduringcelldifferentiation
[1–3,28]. With the possibility of creating induced pluripotent stem
(iPS) cells from adult somatic cells [29,30] has come the opportu-
nity to study how and whether reprogramming into pluripotent
stem cells is accompanied by X-reactivation. These studies have
shown that events on the X-chromosome and stem cell fate are
indeed intimately connected. Below, we will focus on events sur-
rounding cell differentiation and de-differentiation and the fate of
the X-chromosome in ES and iPS cells, specifically those involving
noncoding genes.
notfocusonimprintedX-
2. Mouse X-chromosome regulation
2.1. Mouse ES cells
For random XCI studies, mouse ES cells [31] have served as a
powerful model system and enabled elucidation of function for
many ncRNAs during this process. In undifferentiated female mES
cells where parental epigenetic marks have been erased to be
reprogrammed, both Xs remain active with very low levels of Xist
expression.CelldifferentiationthentriggersXCI,initiatedwithXist
RNA upregulation on the future Xi. Although how Xist is regu-
lated has yet to be fully understood, many studies have established
the 40-kb Tsix ncRNA as a major regulator that antagonizes Xist
induction in cis: deleting Tsix causes hypertranscription of Xist
[19,20,27,32],andoverexpressionofTsixRNApreventsXistupregu-
lation [33,34]. Various mechanisms are involved in Tsix-mediated
repression of Xist: (1) Tsix modulates the chromatin state of Xist
[35–38];(2)itinducesdenovoCpGmethylationandsilencingofthe
Xistpromoter[36,37];and(3)itintersectswiththeRNAimachinery
to silence the Xist promoter [39–41]. Tsix transcription is positively
regulated in cis by Xite, another non-coding gene that functions as
an enhancer of Tsix transcription during mES cell differentiation
[39,42].
While Tsix mediates negative regulation of Xist, a recent study
has revealed another ncRNA, Jpx, in the role of Xist activation [16].
Fig. 1. Xist regulation by the core pluripotency factors. Oct4 and Sox2 bind the non-
coding Tsix and Xite loci, upregulating the expression of Tsix. Xist levels are also
controlled by direct binding of Oct4, Sox2, and Nanog to Xist intron 1.
Like Xist and Tsix, Jpx resides in the Xic [43–45] and is developmen-
tally regulated, showing a 20- to 30-fold increase in its expression
level prior to the initiation of XCI [16]. Deleting Jpx results in two
majorproblems:defectiveXCIandfemale-specificlethality,specif-
ically during differentiation of mES cells. Xist expression is severely
attenuated in female mES cells, and embryoid body formation is
disrupted during cell differentiation. However, the deletion has no
effect in male mES cells, suggesting an essential and direct role
for Jpx in the XCI process. Jpx RNA knockdown experiments using
shRNAs recapitulates the deletion phenotype, thereby implicating
Jpx RNA in the activation of Xist. Unlike other noncoding genes
of the Xic, the Jpx deletion can be rescued by autosomal expres-
sion of a Jpx transgene, which implies that Jpx functions in trans
as a diffusible ncRNA. Finally, truncating Tsix RNA in a Jpx dele-
tion background also rescues Xist expression, indicating that the
two regulatory ncRNAs work in parallel and antagonistic pathways
to control Xist. Thus, Xist RNA levels during XCI are directly regu-
lated by two ncRNA switches, Jpx and Tsix, which help designate
the future Xi and active X (Xa) chromosomes.
Xist ncRNA accumulation on the Xi is almost immediately fol-
lowedbytherecruitmentofPolycombrepressivecomplex2(PRC2)
to catalyze H3K27me3 [13–15]. The search for mechanisms of
PRC2 recruitment to the Xi led to identification of a novel ncRNA
located within the 5?end of Xist called RepA [12]. The 1.6-kb RepA
is an independent transcription unit embedded within Xist that
shares Repeat A, a conserved motif known to be important for X-
chromosomesilencing[17,46].RNAimmunoprecipitation(RIP)and
RNAelectrophoreticmobilityshiftassay(EMSA)revealedthatRepA
directly interacts with Ezh2, a catalytic subunit of PRC2, via a sec-
ondary structure within Repeat A [12]. Autosomal RepA transgenes
couldincreaserecruitmentofPRC2uponinduction,suggestingthat
RepA RNA is sufficient to recruit PRC2 to chromatin. Unlike Xist
RNA, RepA is expressed prior to XCI, and its levels are not upreg-
ulated during cell differentiation. RepA RNA exhibits important
functions in the pre-XCI state, where it plays a pivotal role in de
novo recruitment of PRC2 to the Xic, perhaps aiding in the activa-
tion of Xist [12,17] and enabling progression from pluripotency to
differentiated cell states.
Studies using mES cells have continued to yield novel insights
into the molecular circuitry that links XCI to pluripotency. Recent
findings regarding the pluripotency factor Oct4 have uncovered its
role as a master regulator of X-chromosome counting and pair-
ing [47]. In mES cells, Oct4 directly binds the Tsix and Xite loci
(Fig. 1), proximal to sites occupied by another regulator of X-
chromosome pairing, Ctcf, which physically interacts with Oct4.
A second pluripotency factor, Sox2, also directly binds Xite, while
making indirect contact with Tsix through looping interactions
between the Xite and Tsix domains [47]. Furthermore, Sox2 inter-

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acts with Yy1, a Tsix transactivator that regulates XCI choice.
Because Yy1 is known to bind Ctcf [48], while Sox2 interacts with
Oct4 as part of the core transcriptional circuitry that regulates
pluripotency [49], it is likely that a multifactor complex com-
prising of Oct4, Sox2, Ctcf, and Yy1 directs the nascent stages of
X-chromosome inactivation. In undifferentiated mES cells, biallelic
occupancyofthesefactorsisthoughttopromoteexpressionofTsix
RNA, which in turn blocks the action of RepA and Xist RNAs in the
initiation of X-chromosome silencing.
Through its intrinsic developmental specificity, Oct4 triggers
changes in Xic behavior during the process of mES cell differentia-
tion. Loss of Oct4 during cell differentiation is thought to induce
homologous pairing between the two X-chromosomes [47], an
act mediated by Tsix and Xite and associated with the regulatory
steps of X-chromosome counting and choice that occur prior to
the initiation of XCI [50,51]. Knockdown of either Oct4 or Ctcf pre-
vents pairing interactions from occurring [47,52]. Deleting either
Tsix or Xite also interferes with X-chromosome pairing, and inser-
tion of Tsix and Xite sequences into an autosomal locus results
in ectopic pairing between the autosome and an X-chromosome
[51]. These results support the idea that a complex of Oct4, Ctcf,
and Tsix/Xite sequences underlies the pairing interaction between
the X-chromosomes. It is presently unknown whether ncRNAs
transcribed from Tsix and Xite are required for pairing. However,
inhibition of RNA polymerase II by actinomycin D or ?-amanitin
disrupts the pairing interaction. As differentiation proceeds, the
progressive loss of Oct4 may cause dissolution of the complex and
dissociation of the X-chromosomes, which may result in redistri-
butionoftheTsixtranscriptionfactorsCtcf,Oct4,andYy1fromboth
allelestooneallele,duetothehighlycooperativebindingoffactors
[47,51,53]. By this model, the persistent binding of transcription
factors on the Xa allele sustains Tsix expression exclusively on that
chromosome. Interestingly, Oct4 knockdown has also been shown
to result in biallelic Xist expression, indicating misregulation of X-
chromosomecounting[47].Oct4isthusthefirstknowntrans-factor
that regulates X-chromosome counting.
Pluripotency factors also intersect within the gene body of
Xist/Tsix. Nanog binding sites are found within Xist intron 1 (Fig. 1),
and co-occupancy by Oct4 and Nanog can repress Xist expression,
either directly repressing Xist or indirectly repressing it via Tsix,
which overlaps Xist in this region [54]. Nanog-null male mES cells
display elevated levels of Xist RNA with no change in steady state
levelsofTsix.ItisthusproposedthatNanogmayfunctionindepen-
dentlyofTsixasarepressorofXist.InTsix-truncatedmalemEScells,
Nanog remains bound to Xist intron 1 [54]. Of note, Oct4 and Sox2
also remain associated with Xist intron 1 in Nanog-null male mES
cells.InOct4-nullmalemEScells,however,Sox2andNanogbinding
to Xist intron 1 are compromised. Additionally, a small fraction of
Oct4-null male mES cells display Xist upregulation, suggesting that
Oct4 exhibits a more prominent role than Nanog in Xist regulation
and X-chromosome reprogramming.
Together, Tsix, Oct4, and Nanog serve as important regulators of
Xist expression during mES cell differentiation. The idea that Tsix
andOct4mightregulateXistindependentlyissupportedbythefact
that different mES cell differentiation methods affect Xist expres-
sion differentially when Tsix is deficient [55]. When Tsix?CpGmale
mES cells are differentiated in the absence of all-trans retinoic acid
(RA), only a minute percentage of differentiated cells exhibit Xist
clouds. However, in the presence of RA, partial Xist clouds appear
in almost one-third of Tsix?CpGmale mES cells (the Xist clouds are
generally dispersed and do not necessarily result in genic silenc-
ing). The use of RA to differentiate ES cells was shown to accelerate
downregulation of the general pool of Oct4 mRNA, as well as to
accelerate loss of Oct4 binding to Xist intron 1. In the presence of a
functional Tsix allele, however, the use of RA during male mES cell
differentiation does not lead to ectopic Xist cloud formation. These
resultsindicatethatTsixissufficientforproperXistregulation,irre-
spective of Oct4 binding to Xist intron 1. While Tsix serves as the
primary regulator of Xist, Oct4 may compensate for the absence of
Tsix when male mES cells are differentiated without RA, given the
low incidence of ectopic Xist cloud formation. These results indi-
cate that Oct4 and Tsix act in both coordinated and independent
pathways to regulate Xist levels in mES cells.
2.2. Mouse iPS cells
Mouseinducedpluripotentstem(miPS)cellsaregeneratedfrom
somaticcellsthroughectopicexpressionofthetranscriptionfactors
Oct4, Sox2, Klf4, and c-Myc [29]. Interestingly, converting somatic
female cells into miPS cells results in extensive X-chromosome
reprogramming [56]. The Xi in female miPS cells is reactivated,
and Xist expression becomes undetectable upon direct reprogram-
ming (Fig. 2). Tsix and X-linked gene expression become biallelic,
while Xite is also expressed. The reactivated X-chromosome loses
H3K27me3 and Polycomb group protein enrichment, creating
a transcriptionally permissive chromatin environment. Further-
more, the Xist, Tsix, and Xite ncRNAs are reprogrammed to their
pluripotent, mES-like expression state. When induced to differ-
entiate, miPS cells behave equivalently to mES cells with respect
to XCI: Tsix RNA is downregulated, Xist RNA is upregulated and
cytologically coats one X-chromosome, and the Xi is decorated
by Polycomb proteins and the hallmark H3K27me3 modifica-
tion [13,14]. These findings further underscore the tight linkage
between X-chromosome state and stem cell pluripotency.
During the induction of pluripotency by defined factors, X-
chromosome reactivation is a late event in the reprogramming
process [57]. Epigenetic reprogramming of the X-chromosome is a
hallmark of bona fide female miPS cells, along with the reactivation
of endogenous pluripotency genes and telomerase. Sox2 and Oct4
are reactivated with faster kinetics than the silent X-chromosome,
although these processes are also considered relatively late events
during the reprogramming process. Assessment of endogenous
Sox2 reactivation after ∼18 days of fibroblast reprogramming indi-
cates that a majority of cells express Sox2, while only a small
fraction of cells at analogous time points show X-chromosome
reactivation. Expression of endogenous Sox2 and Oct4 may subse-
quently facilitate silencing of Xist expression through Tsix and Xite
activation, as well as direct binding to Xist intron 1. The molec-
ular mechanism underlying X-chromosome reactivation during
direct reprogramming, however, remains an area for further inves-
tigation, and miPS cells provide an excellent model in which to
investigate the linkage between the epigenetic status of the X-
chromosome and pluripotency.
2.3. Mouse EpiSCs
Mouse pluripotent stem cells are also derived from the epi-
blast layer of post-implantation embryos (d5.5) and are referred
to as epiblast stem cells (mEpiSCs) [58,59]. The epigenetic state
of mEpiSCs differs from mES cells, with their pluripotency signal-
ing pathways, cellular morphology, and gene expression patterns
being more analogous to human ES cells [60]. mEpiSCs express the
pluripotency factors Oct4, Nanog, and Sox2 and differentiate into
the three germ layers, but grow as monolayer colonies in a simi-
lar manner to human ES (hES) cells. Unlike mES cells, mEpiSCs are
Rex1-negative, express FGF-5 and Nodal, and fail to incorporate
into pre-implantation embryos. mEpiSCs have already undergone
XCI (Fig. 2), as evidenced by H3K27me3 on the Xi and reminis-
cent of many hES cell lines (discussed in Section 3.1). Of note, Klf2,
Klf4, and Klf5 levels are downregulated in mEpiSCs when com-
pared to mES cells [61], suggesting that Klf family members might
be involved in X-chromosome reprogramming. Adding exogenous

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Fig. 2. X-chromosome state in mouse pluripotent stem cells. Naïve mES and miPS cells represent the ground state of pluripotency, as evidenced by the presence of two active
X-chromosomes and the absence of Xist RNA. Primed mEpiSCs have already undergone X-chromosome inactivation and represent a developmentally more advanced state.
Klf4 to mEpiSCs, however, fails to induce X-chromosome reacti-
vation and convert these cells into mES cells [62]. Interestingly,
culturing mEpiSCs with exogenous Klf4 in the presence of LIF and
small molecule inhibitors of Mek/Erk MAP kinase signaling and
glycogen synthase kinase (2i) induces their transition to a mES-like
state, along with epigenetic reprogramming of the X-chromosome
and efficient chimeric contribution [62]. The efficiency of convert-
ingmEpiSCstomEScellsusingthisprocedureis∼0.1%,comparable
to the efficiencies observed when reprogramming fibroblasts to
miPS cells using defined factors (Oct4/Sox2/c-Myc/Klf4) [63]. In
part, low efficiencies may stem from the potential requirement
for stochastic events to facilitate the epigenetic reprogramming
process [64].
3. Human X-chromosome regulation
3.1. Human ES cells
Assessing XIST ncRNA and XCI status in hES cells provides a
measure of their epigenetic stability, which is an important con-
siderationfortheirpotentialapplicationsinregenerativemedicine.
Existing hES cell lines exhibit diverse patterns of XIST expression,
indicativeofbothpre-andpost-XCIstates[65–70].Becauseofsimi-
laritiesbetweenmEpiSCsandhEScells(morphology,Activin/Nodal
signaling for pluripotency, bFGF growth requirements), it was
hypothesized that hES cells represent post-XCI cells, similar to
mEpiSCs. Numerous female hES cell lines have been extensively
characterized with respect to XCI, revealing the existence of three
distinctclassesofXCIstatus(Fig.3)[70,71].LinesofhEScellsdesig-
natedas‘ClassI’showrelativelylowlevelsofXISTexpressioninthe
undifferentiatedstate,andupondifferentiation,XISTisupregulated
and the number of cells with large XIST RNA nuclear foci increases.
In ‘Class II’ lines, XIST expression levels are comparable in both the
undifferentiated and differentiated states, indicating that XCI has
already occurred in these hES cell lines. ‘Class III’ lines no longer
express XIST ncRNA, whether in the undifferentiated or differen-
tiated state, yet maintain monoallelic X-linked gene expression,
suggestive of an aberrant epigenetic state. Thus, Class III hES cells
appear to have undergone XCI, suggesting that XIST expression
was lost after XCI was established. While expression of X-linked
repetitive elements remain mostly suppressed, there may be par-
tial reactivation of a small number of X-linked genes [66,69,70]. Of
note, Classes I and II cells readily transition into Class III cells with
prolongedculture,demonstratingthehighlydynamicandunstable
nature of the epigenetic state in hES cells (Fig. 3) [70].
3.2. Naïve human ES cells
Currentresearcheffortsareaimedatestablishingandmaintain-
ing hES cells in conditions that would enhance their epigenetic
stability. A recent study hypothesized that replicating physiolog-
ical oxygen concentrations of the early embryo (hypoxic; 5% O2)
would be beneficial for the derivation of epigenetically stable hES
cells [68]. Of note, XCI status was the most sensitive measure for
distinguishing between lines generated and maintained in either
ambient or hypoxic oxygen concentrations. Importantly, hES cells
in a pre-XCI state were only generated when early embryos and
dissected ICM cells were cultured at physiological oxygen levels.
Using physiological O2concentration resulted in the derivation of
developmentally ‘naïve’ [72] hES cells that display two active X
chromosomes [68] (Fig. 3), as indicated by the use of XIST and
XCI markers as diagnostic tools. These lines upregulated XIST upon
differentiation and formed cytologically visible XIST clouds, sug-
gesting that naïve hES cells in fact resemble mES cells instead of
mEpiSCs. hES cells derived in ambient oxygen had already under-
gone XCI, similar to the majority of hES cells derived previously.
Another recent study, however, isolated pre-XCI Class I hES cells in
ambient oxygen, but these lines quickly became Classes II and III
lines within 15–20 passages [71]. Both studies observed extensive
DNA methylation at the XIST promoter in pre-XCI lines, sugges-
tive of XIST silencing on both alleles. hES cells cultured in ambient
O2conditions (20%), however, displayed ∼50% reduction in XIST
promotermethylation,alongwithactivationofoneXISTallele,indi-
cating their conversion to Class II status. Class III hES cell lines
grown in ambient oxygen continued to display monoallelic X-
linked gene expression despite the loss of XIST RNA. Although loss
of XIST RNA in Class III hES cells resulted in varying degrees of
X-linked gene derepression, genes that remain silenced might be
repressed by H3K27me3, which was present on the Xi despite the
absenceofXIST[68][note:H3K27me3fociarenotobservedinClass
III hiPS and hES cells [70,73]].
Thus, naïve hES cells (Class I) convert to the primed state (Class
II) upon exposure to hyperoxic conditions, undergoing irreversible
XCI. Additionally, harsh freeze-thaw cycles, as well as treatment of
naïve hES cells with compounds that induce cellular stress, result
in XIST gene activation, indicating that naïve hES cells are acutely

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Fig. 3. X-chromosome state in human ES cells. Deriving hES cells under physio-
logical O2(5%) generates hES cells that are more likely to be Class I, lacking XIST
expression and other markers of XCI (‘naïve’), whereas derivation under hyperoxic
conditions more likely yields hES cells of the Class II type, having already undergone
XCI (‘primed’). However, ambient oxygen levels can also yield Class I cells. Cellular
stress converts Class I and Class II hES cells into Class III.
sensitive to various types of cellular stress. Treatment of naïve hES
cellswithantioxidantsconfersprotectionagainstXCIandXISTacti-
vation when cells are exposed to hyperoxic conditions, indicating
that oxidative stress is a key determinant of precocious XCI in the
undifferentiated, naive state.
Another study recently showed that HDAC inhibitors help
promote epigenetic stability and decrease cellular differentiation
during routine culture of hES cells [76]. Treatment of the H9
female hES cell line, containing a mixed population of XIST+ and
XIST− cells, resulted in complete loss of XIST RNA in the undif-
ferentiated state, with XIST upregulation being observed during
cell differentiation. HDAC inhibitor treatment, which reversibly
alteredexpressionofseveralhundredgenesandincreasedcellcycle
growth, also reversed the XCI state and rendered this cell line more
Class I-like [76]. However, since the authors only examined one
female hES cell line known to have epigenetic variability between
differentpassagesandlaboratories,thiseffectmaynotbeuniversal
for all hES cell lines.
It has also been proposed that priming hES cells with a specific
cocktail of small molecules and transgenes carrying pluripotency
factorscanconvertepigeneticallyabnormalhEScellsintonaïvehES
cells [74]. Specifically, adding ERK1/2 inhibitor PD0325901 (PD),
GSK3 inhibitor CHIR99021 (CH), and Klf4/Klf2 regulator Forskolin
[75],aswellasprovidingOct4andKlf4expression,convertsprimed
hES and human induced pluripotent stem (hiPS) cells into a naïve
state after ∼10 days in culture (Fig. 3). Converted Class I hES cells
lack XIST clouds in the undifferentiated state, and unlike their
parental Class II state, can be passaged as single cells with trypsin,
similar to mES cells and without the acquisition of chromosomal
abnormalities.Moreover,convertednaïvehEScellsexhibitbiallelic
XIST promoter methylation, indicating XIST silencing, and display
global gene expression patterns that are characteristic of naïve
mES cells. Upon differentiation, naïve hiPS cells upregulate XIST
RNA, which coats the Xi (experiments were carried out in ambient
oxygen conditions; differentiated naïve hES cells were not exam-
ined). Given the inherent epigenetic instability of hES cells, defined
small molecule cocktails might help stabilize the naïve hES-state.
Interestingly, global gene expression patterns for naïve hES and
hiPS cells resemble those of mES cells, suggesting that these naïve
human cell types might serve as true representations of the inner
cell mass in human embryos.
These studies highlight the permissiveness of X-chromosome
epigenetic reprogramming in established hES cell lines using
defined culture conditions, while also highlighting the usefulness
ofncRNAmarkersinthestudyofregenerativemedicine.WhileXIST
ncRNA now appears to be an excellent marker for determining epi-
genetic stability in hES cells, additional studies are needed to learn
about other ncRNAs that regulate XCI, particularly those that have
been shown to play crucial roles in the mouse system. Given that
early mouse and human development share more similarities with
respect to XCI than previously thought, it is likely that human TSIX
[77]andJPX[78],bothwithunknownfunctionsinpluripotentcells,
might also be harnessed as diagnostic tools for assessing hES cell
epigenetic stability.
3.3. Human iPS cells
Studies of hiPS cells are currently being pursued with great
interest, given their enormous potential in personalized regener-
ative medicine [79]. The ectopic expression of Oct4, Sox2, Klf4,
and c-Myc in somatic cells yields hiPS cells with high degrees of
molecular and functional similarities to hES cells [80]. Assessing
the X-reactivation state provides valuable insight into the epi-
genetic status of hiPS cells and indicates whether acquisition of
the pluripotent ground state has been achieved. Two recent stud-
ies on hiPS cells examined whether induction of pluripotency
using defined factors resulted in epigenetic reprogramming of
the X-chromosome, leading to different conclusions. One study
found that hiPS cells, generated using either lentiviral or retroviral
reprogramming vectors, exhibited similar genome-wide expres-
sion profiles to hES cells, while forming teratomas in vivo that
containedcelltypesfromallthreegermlayers[73].Bothlentiviral-
and retroviral-derived Nanog-positive hiPS cells exhibited an
X-chromosome coated with XIST RNA (Class II) in ∼88% of NANOG-
positive cells (Fig. 4) – an unanticipated result given prior findings
that X-reactivation accompanies reprogramming into iPS cells in
the mouse system. The XIST-coated X-chromosomes were also
transcriptionally silent, evidenced by the lack of X-linked gene
expression. All cells within a selected hiPS cell clone exhibited XCI
on the same X-chromosome, indicating that a single fibroblast had
clonally expanded to generate the hiPS cell line without undergo-
ing X-reactivation. In control fibroblasts, the Xi was coated with
H3K27me3, but not the Polycomb group proteins that mediate this
histone mark. However, in fibroblast-derived hiPS cells, the Xi dis-
played enrichment for Polycomb group proteins, but only after
endogenous NANOG has been activated. Contrary to these findings,