Residual Expression of Reprogramming Factors Affects
the Transcriptional Program and Epigenetic Signatures
of Induced Pluripotent Stem Cells
Cesar A. Sommer1., Constantina Christodoulou2., Andreia Gianotti-Sommer1., Steven S. Shen3, Badi
Sri Sailaja4, Hadas Hezroni4, Avrum Spira3, Eran Meshorer4, Darrell N. Kotton2,5*,
1Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America, 2Boston University
Pulmonary Center, and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America, 3Section of Computational
Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America, 4Department of Genetics, Institute of Life
Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel, 5Center for Regenerative Medicine (CReM), Boston University School
of Medicine, Boston, Massachusetts, United States of America
Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to
generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an
invaluable resource for medical research and drug development. Residual reprogramming transgene expression from
integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced
developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and
after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent
transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors
prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression
profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones
show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we
show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we
find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2ONvs Gtl2OFFiPSC clones. Our
findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of
any residual transgene expression.
Citation: Sommer CA, Christodoulou C, Gianotti-Sommer A, Shen SS, Sailaja BS, et al. (2012) Residual Expression of Reprogramming Factors Affects the
Transcriptional Program and Epigenetic Signatures of Induced Pluripotent Stem Cells. PLoS ONE 7(12): e51711. doi:10.1371/journal.pone.0051711
Editor: Atsushi Asakura, University of Minnesota Medical School, United States of America
Received August 10, 2012; Accepted November 5, 2012; Published December 14, 2012
Copyright: ? 2012 Sommer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: GM and DNK are supported by NIH PO1 HL047049-16A1, 1RC2HL101535-01, and 1R01 HL095993-01. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (GM); email@example.com (DNK)
. These authors contributed equally to this work.
The discovery that differentiated adult cells can be repro-
grammed to a state of pluripotency through the introduction of
a defined set of transcriptional regulators has opened new avenues
for understanding and treating degenerative diseases . Patient-
specific induced pluripotent stem cells (iPSCs) offer a unique
opportunity to develop personalized regenerative medicine ther-
apies because they lack the ethical issues associated with
embryonic stem cells (ESCs). Despite the excitement and promise
surrounding iPSCs, researchers are just beginning to elucidate the
molecular mechanisms that operate during and after the induction
Reprogramming was originally achieved via retroviral transfer
of Oct4, Klf4, Sox2 and c-Myc (OKSM) , but this approach
was later associated with a high risk of tumor formation due to
spontaneous reactivation of transgenes . Further attempts to
increase the safety of the technique led to the removal of c-Myc
from the transcription factor cocktail and the development of
excisable vectors as well as non-integrating gene delivery
methodologies based on adenoviruses, plasmids, protein and
RNA, reviewed in [4,5]. Yet, because of its simplicity, high
efficiency and reproducibility, a large number of iPSC lines have
been generated with retroviruses/lentiviruses [6,7,8,9], and viral
transduction remains widely used. Indeed, the majority of iPSC-
based disease-modeling studies reported thus far have relied on
transgene-carrying iPSC lines [10,11]. Residual expression of the
integrated viral transgenes in the reprogrammed cells, neverthe-
less, has been shown to affect their biological properties both in vivo
and in vitro [12,13]. In this context, it is important to evaluate the
overall gene dysregulation caused by the presence of the
transgenes if transgene-carrying iPSCs are to be employed for
drug screening, tissue development or disease modeling. However,
efforts to gain further insight into this phenomenon have been
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hampered by the presence of multiple copies of the viral
transgenes in the iPSC clones, which often exhibit different
degrees of silencing . As a result, the extent to which persistent
expression of the reprogramming factors perturbs the transcrip-
tional program of iPSCs has not been systematically assessed.
Furthermore, it remains unclear whether iPSCs derived with or
without c-Myc differ when comprehensively compared by global
gene expression profiling.
More recent reports have raised additional controversies
regarding subtle genetic and epigenetic differences between iPSCs
and ESCs that arise during reprogramming [15,16,17,18],
although some of these could be due to lab-specific effects
[19,20]. Notably, the epigenetic status of a single imprinted region,
the Dlk1-Dio3 gene cluster, seems sufficient to predict the
developmental potential of mouse iPSCs [21,22]. For example,
iPSC clones exhibiting epigenetic silencing of Gtl2, a member of
the Dlk1-Dio3 cluster normally expressed from the maternally
inherited allele, contribute poorly to chimeras and fail to produce
viable mice through tetraploid complementation. To date, it has
not been clear whether aberrant Gtl2 silencing in iPSCs results
from the selection of a subset of previously mis-imprinted parental
fibroblasts or occurs at some point during the reprogramming
process. Understanding which of these differences are introduced
during reprogramming and whether they are functionally relevant
is critical since they may influence potential downstream
We previously described the development of the STEMCCA
(‘‘STEM Cell CAssette’’) polycistronic lentiviral vector for the
efficient generation of iPSCs [23,24]. This vector was further
modified by the insertion of a lox-P site, providing a way to derive
transgene-free iPSCs from both mouse and human somatic tissues,
such as fibroblasts [8,13] and peripheral blood cells . Here we
employ STEMCCA a) to systematically characterize the tran-
scriptional profile of mouse iPSCs before and after excision of
a single copy of the reprogramming cassette, b) to compare iPSCs
generated with 3 vs 4 factor reprogramming methodologies, both
before and after reprogramming factor withdrawal, and c) to
quantify the frequency and kinetics of aberrant Dlk1-Dio3 locus
imprinting in iPSCs. We demonstrate that transgene removal
attenuates gene expression differences between iPSCs and ESCs
and that cells reprogrammed with and without c-Myc are
indistinguishable by microarray analysis. In addition, we provide
evidence that exogenous expression of Klf4 results in augmented
binding of Klf4 to the promoter region of Gtl2, which might affect
the observed silencing of this locus during reprogramming. Finally,
we confirm that iPSCs retain the ability to differentiate towards
the hepatic lineage regardless of the epigenetic status of this locus.
Distinctive Gene Expression Profiles Characterize iPSCs
before and after Excision of a Constitutively Expressed
To gain insight into the transcriptome changes that result from
the removal of exogenous reprogramming factors we performed
genome-wide gene expression analysis on iPSC lines before and
after Cre recombinase-mediated deletion of the STEMCCA
polycistronic vector. Ten iPSC clones were derived from Sox2-
GFP knock-in postnatal mouse fibroblasts using either the EF1a-
STEMCCA-loxP vector (N=5). These vectors allow for the co-
expression of three (OKS) or four (OKSM) factors respectively, as
previously described . To minimize genome modification and
allow for a proper comparison across the iPSC lines we selected
(N=5) orthe EF1a-
clones harboring single proviral integrations and performed Cre-
recombinase treatment in order to obtain a ‘‘transgene-free
version’’ for each of the 10 clones. Successful excision of the stem
cell cassette was confirmed by Southern blot analysis (Figure S1 A).
As previously reported, the iPSC lines generated with STEMCCA
exhibited expression of pluripotency markers and were able to
form teratomas after transplantation into immunodeficient mice
(Figure S1 B). In addition, all clones displayed a normal karyotype
(Figure S1 C) and the proliferation properties of ESCs (data not
The different groups of iPSC clones that were subjected to
microarray analysis along with five ESC subclones obtained from
a Sox2-GFP ESC line are shown in Fig. 1A. To avoid introducing
transcriptional changes that could be due to residual gene
expression of the donor cells  or extended culturing , all
the iPSC clones were profiled at passages p15-18. We performed
Principal Components Analysis (PCA) on the whole set of 25
samples, which revealed clear segregation of ESCs, transgene-
carrying iPSCs (OKS and OKSM) and transgene-free iPSCs
(OKS-Cre and OKSM-Cre) into three distinct clusters (Fig. 1B).
PCA was unable to distinguish three- and four-factor iPSCs;
instead it was revealed that the presence of trangenes was the
major source of the observed variation. Thus, transgene-carrying
iPSCs and transgene-free iPSCs are characterized by unique gene
expression patterns. Notably, a more detailed examination of the
gene expression profiles revealed two apparent subgroups within
the transgene-carrying iPSC cluster, with the 5 clones exhibiting
higher levels of the polycistronic transcript (OKSM-D, E, F and
OKS- 6,15; Fig. 1C) located farther away from the ESC control
group along principal component 1 (Fig. 1B). Thus, the degree of
transcriptional dysregulation in transgene-carrying iPSCs appears
to be correlated with residual transgene activity and relatively
small (1.5- to 2.5-fold) increases in exogenous reprogramming
factor expression are sufficient to elicit genome-wide transcrip-
tional changes that can be identified by PCA.
Global Gene Expression Differences between iPSCs and
ESCs are Attenuated Following Transgene Excision
We performed a two-way ANOVA of the 25 samples to
determine: a) the effect of transgene removal on the global
transcriptome of iPSCs (Cre effect), and b) differences in gene
expression between ESCs, 3 factor iPSCs and 4 factor iPSCs (cell
effect). Comparing the datasets of pre-Cre and post-Cre iPSCs we
found 2,327 significantly differentially expressed probesets (FDR-
adjusted p-value ,0.01; see complete gene list in Table S1).
Hierarchical clustering using this subset of genes revealed distinct
expression patterns specific to each of the three groups and
increased similarity of iPSCs to ESCs following transgene excision
(Fig. 1D and Figure S2). Importantly, we confirmed that the total
levels of the reprogramming factors were significantly increased in
all 10 transgene-carrying iPSC clones. However, the endogenous
levels were similar across the iPSC lines, both before and after
Cre-mediated excision (Figure S3), consistent with the reactivation
of endogenous pluripotency-associated genes in iPSCs repro-
grammed with STEMCCA . Our results demonstrate that the
presence of residual reprogramming transgenes significantly affects
the global transcriptome profile of iPSCs and deletion of the
reprogramming transgenes brings iPSCs transcriptionally closer to
To investigate the molecular changes brought about by the
excision of STEMCCA the list of differentially expressed genes
was uploaded into the online functional annotation tool DAVID.
Gene ontology analysis indicated a number of significantly
enriched GO terms that corresponded to metabolic and bio-
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Figure 1. Global gene expression differences between iPSCs and ESCs are attenuated following excision of a lentiviral
reprogramming cassette. (A) Schematic representation of the different groups of iPSC/ESC lines subjected to microarray analysis. iPSCs carrying
a single copy of a ‘‘floxed’’ STEMCCA vector encoding either three (OKS) or four (OKSM) reprogramming factors were treated with Cre-recombinase to
generate transgene-free OKS-Cre and OKSM-Cre iPSC clones. Five subclones of the Sox2-GFP/M2rtTA ESC line were isolated, expanded and included
Gene Dysregulation in Transgene-Carrying iPSCs
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synthetic processes, tissue development, and morphogenesis (Table
S2). Most importantly, among the over-represented functional
categories were those related to chromatin assembly and
epigenetic regulation of gene expression. Genes in these categories
included DNA modifiers (DNMT3A, MAEL), chromatin binding
proteins (MBD4, MBD1, PIWIL4), chromatin remodelers (HAT1,
SIRT6, SIRT7, SUV39H1, SMARCC1, MYST3), and members
of the histone family of proteins. Most of these genes displayed
levels similar to ESCs following transgene excision (see Table S3
for a complete list of genes and log2 values). Some of these
regulators participate in chromatin remodeling complexes that are
required for the establishment and maintenance of the pluripotent
state [27,28] and might coordinate the epigenetic changes that
accompany reprogramming . In addition, among the sixteen
KEGG pathways identified by DAVID as significantly altered in
the gene set (EASE score ,0.05; Table S4), the TGF-beta
signaling pathway was one of the most enriched (EASE
score=0.01; fold enrichment=1.97). Notably, many of the
differentially regulated genes that mapped to this pathway,
including Smad2/3, Smad4, Smad7, Id2, Id4, and activin A
receptor type 1, displayed a shift in their expression values towards
the levels observed in ESCs (Table S5). These differences provide
a possible biological explanation for the diminished in vivo
developmental competence of transgene-carrying iPSCs as well
as their poor response to Activin A stimulation in vitro .
Collectively, our findings are in agreement with previous
observations  and suggest that residual transgene expression
prevents iPSCs from assuming the complete genetic program
associated with ESCs.
iPSCs Generated with and without cMyc are
Indistinguishable by Gene Expression Profiling
Recent data indicates that the transcription factor c-Myc acts
mainly during the early stages of reprogramming by inducing cell
cycle changes consistent with self-renewal and/or promoting
dedifferentiation . An additional, more direct role of c-Myc in
the establishment of pluripotency would be possible through its
ability to recruit chromatin modifiers [31,32,33]. We reasoned
that reprogramming in the presence or absence of exogenous c-
Myc could result in iPSCs exhibiting similar, but not necessarily
identical, gene expression patterns. Therefore we compared the
genome-wide datasets of iPSCs generated with and without c-Myc.
In contrast to the substantial Cre-effect on gene expression, we
found zero genes were differentially expressed when comparing
iPSC clones generated with 3 factor vs 4 factor reprogramming
(cell effect; Figure S2) (FDR-adjusted p-value ,0.1). We conclude
that iPSCs generated with and without c-Myc have virtually
indistinguishable gene expression patterns.
Residual Transgene Expression may Influence Epigenetic
Silencing of the Imprinted Gtl2 Gene during
Recent studies have reported that the conserved imprinted
Dlk1-Dio3 region on mouse chromosome 12qF1 is actively
transcribed in fully pluripotent iPSCs but silenced in iPSC clones
that lack the capacity to support the development of ‘‘all-iPSC
mice’’ [21,22]. In particular, the maternally expressed gene Gtl2,
a member of this cluster that is active in ESCs, was found
aberrantly silenced in most iPSC clones despite being normally
imprinted (,50% CpG methylation) in the starting fibroblast
To gain insight into the epigenetic regulation of the Dlk1-Dio3
domain during reprogramming we first analyzed the expression
values of Gtl2 in our microarray datasets but were unable to detect
statistically significant differences in expression levels between
ESCs and iPSCs by ANOVA (data not shown). Therefore we
determined Gtl2 mRNA expression levels in the 20 iPSC clones via
qRT-PCR. We identified two of 20 iPSC clones (5-Cre and 15-
Cre) with low Gtl2 expression levels compared to control ESCs,
suggesting these clones were ‘‘Gtl2OFFclones’’, possibly due to
aberrant silencing of the imprinted Dlk1-Dio3 locus, as has been
previously reported . Surprisingly Gtl2 mRNA expression was
easily detected at levels similar to or above the control ESCs in the
majority of iPSC clones, suggesting they were ‘‘Gtl2ONclones’’
(Fig. 2A). Based on previously published data  we speculated
that the differences in the observed Gtl2 mRNA expression in
iPSCs would be correlated with differences in the methylation
status of two differentially methylated regions (DMR) of the Dlk1-
Dio3 locus: the promoter DMR (Gtl2-DMR) as well as the
intergenic region located between the Gtl2 and the Dlk1 gene (IG-
DMR). We identified low (,30%) or normal (,60%) methylation
in both IG-DMR as well as Gtl2-DMR of the Dlk1-Dio3 locus in
10 out of 10 transgene-carrying iPSC clones. Cre-recombinase
treatment and removal of the overexpressing transgenes was
accompanied by changes in the methylation status of IG-DMR
and Gtl2-DMR. We identified that 2 clones remained hypo-
methylated (OKSM-Cre D and OKS-Cre 11), 6 clones displayed
normal methylation levels (OKSM-Cre B, C, E, F and OKS-Cre
6, 10), and two clones OKS-Cre 5 and 15 became hypermethy-
lated, indicating that aberrant imprinting of these 2 Gtl2OFFclones
occurred following reprogramming factor withdrawal (Fig. 2A).
The high frequency of normally methylated clones contrasts with
previous studies which have suggested that the majority of mouse
iPSC clones are mis-imprinted [22,23]. Importantly, the Sox2-
GFP tail-tip fibroblasts (TTFs) that were used to derive the 20
iPSC clones not only expressed very high levels of Gtl2 mRNA but
also showed normal methylation status at the IG-DMR and Gtl2
DMR of the Dlk1-Dio3 locus.
One difference in the reprogramming methodology used to
generate the 20 iPSC clones in this study, compared to previously
published iPSCs is the use of constitutively active vs. dox-inducible
reprogramming methods [22,23]. Thus, we sought to determine
whether the duration of reprogramming and/or the reprogram-
ming system could be responsible for the previously observed high
frequency of aberrantly imprinted iPSC clones. Hence, we
performed a parallel reprogramming experiment using either
constitutive or inducible reprogramming vectors and short vs long
durations of transgene overexpression. R26-M2rtTA TTFs were
isolated from a male R26-M2rtTA knock-in mouse, followed by
as a control. (B) Principal Components Analysis (PCA) performed on the microarray datasets clearly separates ESCs, transgene-carrying iPSCs (OKS and
OKSM) and transgene-free iPSCs (OKS-Cre and OKSM-Cre) into three distinct groups, indicative of similar but distinctive gene expression profiles.
Notably, PCA is unable to discriminate iPSCs generated with 3 or 4 factors, both before and after transgene removal. Instead, the presence of the
transgenes appears to be a major factor influencing the iPSC transcriptome. (C) qRT-PCR measurement of the residual transcriptional activity of the
reprogramming vector demonstrates differences in expression across the iPSC lines that correlate with the degree of gene dysregulation revealed by
PCA. (D) Hierarchical clustering of the 2,327 genes significantly different between transgene-carrying iPSCs and transgene-free iPSCs (two-way
ANOVA, FDR-adjusted p-value ,0.01) reveals distinct patterns of gene expression specific to each of the three groups and increased similarity of
iPSCs to ESCs following transgene excision.
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transduction with either Tet-STEMCCA (inducible OKSM) or
Ef1a-STEMCCA (constitutive OKSM). TTFs transduced with
inducible OKSM were exposed to doxycycline for either 12 days
(clones A1-4), 20 days (clones C1-4), or for the entire duration of
the experiment (clones B1-4). All iPSC clones were picked on day
20. Four additional iPSC clones generated by constitutive OKSM
were also picked on day 20 and treated with Cre recombinase to
remove the reprogramming cassette. All 20 clones were passaged
18 times to ensure stability of colony morphology prior to
harvesting RNA and genomic DNA. Surprisingly, all 20 clones
deriving from this strain of TTFs were found to exhibit
hypermethylation of the IG-DMR and Gtl2 DMR (Fig. 2B). In
concordance with the hypermethylated state of this locus, Gtl2 and
Rian, genes that are typically transcribed from the maternally
inherited allele, were expressed at low levels in all clones (Fig. 2B).
In contrast, Dlk1, a gene typically expressed from the paternally
inherited allele of the Dlk1-Dio3 locus was expressed in all iPS
clones at levels similar to the ESC control cells (data not shown).
Moreover, the 20 iPSC clones derived from the R26-M2rtTA
TTFs did not display any methylation changes before and after
removal of the reprogramming transgenes as previously observed
in the 20 iPSC clones derived from Sox2-GFP TTFs (summarized
in Table S6). Our overall results indicate an overall frequency of
Dlk1-Dio3 aberrant imprinting in 63% (14/22) of iPSC clones
following reprogramming transgene withdrawal, and the frequen-
cy of misimprinting does not appear to correlate with the
constitutive vs. inducible vector system we employed. In addition,
these findings suggest that the genetic background of the somatic
cells may to some extent explain the variability in the frequencies
of mis-imprinted iPSC clones. Specifically, reprogramming of
C57BL/6 fibroblasts gave rise to hypermethylated Gtl2OFFiPSC
clones, while the same experimental approach yielded mostly
Figure 2. Epigenetic status and transcriptional activity of the Gtl2 locus in iPSC lines generated with STEMCCA. (A) Gtl2 transcript
levels were estimated by qRT-PCR in the starting cell population (TTFs), ESCs, and the 20 iPSC clones profiled by microarray. The percentage of
methylated CpG dinucleotides in the Gtl2 IG-DMR and Gtl2 DMR were determined by pyrosequencing of sodium bisulfite-treated genomic DNA.
Asterisks indicate Gtl2OFFiPSC clones. (B) Twenty additional iPSC clones were derived from TTFs isolated from a different mouse strain (C57BL/6),
using either the Doxycycline (Dox)-inducible or the constitutive STEMCCA vector and analyzed as in (A). Doxycycline was withdrawn at the indicated
time points or kept throughout the expansion of iPSCs. n.d.: not detected.
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clones with low to normal Gtl2 methylation levels when C57BL/
66129/sv fibroblasts were transduced.
Klf4 is Recruited to the Gtl2 Promoter Region during
Reprogramming to Pluripotency
The observation that some of the iPSC clones exhibited
hypomethylated DMRs compared to TTFs before transgene
withdrawal, suggested that enforced expression of the reprogram-
ming factors may delay and/or inhibit the acquisition of epigenetic
marks in this region. For example, it has been demonstrated that
Klf4 can bind to the promoter region of target genes and alter
histone modifications thus regulating gene expression . Hence,
to explore a possible role of the reprogramming factors in the
changes/establishment of the epigenetic status of Gtl2 we
investigated their global DNA binding sites using previously
published ChIP-sequencing (ChIP-seq) data . A significant
peak of tags was observed for Oct4 ,12.5 kb upstream to the Gtl2
transcription start site (TSS), but more importantly, a Klf4-binding
site was identified within the Gtl2 imprinted domain next to the
TSS (Fig. 3A). To verify and quantify Klf4 binding to the
upstream region of Gtl2 we performed chromatin immunoprecip-
itation (ChIP) followed by qPCR (ChIP-qPCR) analysis using an
antibody directed against Klf4. The results, shown in Fig. 3B as
fold-enrichment of Gtl2 relative to the control antibody, confirmed
the binding of Klf4 to the Gtl2 promoter in ESC/iPSCs.
Moreover, sustained ectopic expression of Klf4 in transgene-
carrying iPSCs resulted in increased binding compared to
transgene-free iPSCs, which showed values similar to ESCs
(Fig. 3B). Increased binding of Klf4 was not due to unspecific
binding due to ectopic expression since ChIP for Sox2 did not
show any differential binding between the different cell lines (not
iPSCs Undergo Efficient Differentiation into Hepatocyte
Precursors Despite Differences in the Methylation Status
of Gtl2 DMRs
We have previously reported that aberrantly imprinted iPSC
clones do not show diminished endodermal or hepatic differen-
tiation capacity when compared to control ESCs, despite aberrant
silencing of Gtl2 expression . During those studies we observed
significant induction of Gtl2 in ESCs but not in Gtl2OFFiPSC
clones. However, lack of access to Gtl2ONclones precluded
a syngeneic comparison of OFF vs ON iPSCs. In these studies, we
sought to assess whether sequential differentiation of syngeneic
Gtl2OFFvs Gtl2ONiPSC clones into endoderm followed by early
hepatic lineages would reveal differences in the capacities of these
clones to either induce Gtl2 expression or to induce expression of
marker genes of endodermal and hepatic differentiation. Because
ectopic expression of reprogramming factors negatively impacts
the developmental competence of iPSCs , only transgene-free
clones were evaluated. Consistent with the epigenetic status of the
Gtl2 DMRs in the undifferentiated state, we observed upregulation
of Gtl2 only in ESCs and Gtl2ONiPSC clones in response to
growth factor stimulation (Fig. 4). In contrast, relatively low levels
of Gtl2 transcript were detected in hypermethylated Gtl2OFFclones
regardless of the differentiation state (Fig. 4). Dlk1, the paternally
inherited gene, was upregulated in all iPSC clones during
differentiation. Notably, all the clones showed upregulation of
the endoderm marker Sox17 at day 7 of differentiation and clear
capacity to upregulate hepatic marker genes AFP and albumin
(Fig. 4). These results emphasize that directed differentiation of
iPSCs to lineages that normally exhibit upregulation of imprinted
genes such as Gtl2 further accentuates the differences in Gtl2 gene
expression between normal vs aberrantly imprinted iPSC clones
observed in the basal undifferentiated state. In addition our results
further support the observation that aberrant silencing of Gtl2
Figure 3. Klf4 is recruited to the Gtl2 promoter region during reprogramming to pluripotency. (A) Putative binding sites for the
Yamanaka factors were identified in the Gtl2 promoter region using ChIP-seq data. (B) ChIP-qPCR analysis confirmed increased binding of Klf4 to the
Gtl2 imprinted domain in transgene-carrying iPSCs. Cross-linked protein-DNA complexes were immunoprecipitated from whole cell extracts of ESC/
iPSCs using an anti-Klf4 or a control antibody and DNA within the precipitates was isolated and amplified using primers specific for the Gtl2 promoter.
Results are shown as fold-enrichment relative to the control antibody.
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need not adversely impact the capacity of Gtl2OFFclones to
undergo directed differentiation, as previously published .
Through the systematic comparison of the transcriptional
profiles and epigenetic signatures of iPSCs before and after
excision of a single copy of a polycistronic reprogramming cassette
we have gained novel insights into the molecular events that occur
during iPSC generation. Here, we provide evidence indicating that
i) persistent transgene expression prevents iPSCs from attaining an
ESC-like transcriptional program; ii) iPSCs reprogrammed with
and without c-Myc exhibit highly similar gene-expression profiles;
iii) reprogramming results in aberrant imprinting of the Dlk1-Dio3
locus in some but not all iPSC clones generated with our
polycistronic cassette; and iv) Klf4 binds strongly to the Gtl2
promoter and shows decrease binding upon removal of the
Lentiviral vectors seem to resist methylation-dependent silenc-
ing in mouse pluripotent cells . Consequently, incomplete
transgene silencing following reprogramming has been shown to
negatively affect the differentiation potential of iPSCs, possibly by
antagonizing the transcriptional programs triggered by develop-
mental cues . We found that transgene-carrying iPSCs display
a transcriptional pattern that distinguishes them from transgene-
free iPSCs. Moreover, the degree of transcriptional dysregulation
is correlated with residual transgene activity and relatively small
(1.5- to 2.5-fold) increases in exogenous reprogramming factor
expression appear to elicit genome-wide transcriptional changes
that can be identified by PCA. It should be noted that transgene-
carrying iPSCs have activated the endogenous pluripotency
regulators Nanog and Oct4  and do not represent an
intermediate partially reprogrammed state. Our findings suggest
that rather than being associated with a random perturbation of
the genome, residual expression of the reprogramming factors in
iPSCs induces a genetic program that supports their self-renewal
and their ability to differentiate, albeit at a reduced efficiency
[12,13]. In line with this notion, transcriptional differences
between iPSCs and ESCs are attenuated following withdrawal of
exogenous reprogramming transgenes. Conceivably, these changes
reflect the fine-tuning of the regulatory circuitry underlying
pluripotency that occurs only after exogenous expression of the
Yamanaka factors is withdrawn.
Pluripotency can be induced in the absence of c-Myc over-
expression, albeit at a low efficiency and delayed kinetics [37,38].
Furthermore, the experimental evidence suggests that the main
role of this factor is to suppress somatic cell-specific gene
expression during the initial stages of reprogramming .
Consistent with these observations, we found that the addition of
c-Myc to the reprogramming cocktail has little to no effect on the
transcriptional pattern of iPSCs, even under continuous transgene
expression. Notably, evidence exists that iPSCs produced in the
absence of c-Myc behave differently. For example, OKS-iPSCs
displayed reduced competence for germline transmission com-
pared to OKSM-iPSCs [39,40], but exhibited enhanced in vitro
cardiogenic potential in another study . Apart from residual
expression of c-Myc during differentiation or in vivo development,
the reasons for these differences remain unclear. However,
because c-Myc might participate in chromatin remodeling during
reprogramming , we cannot exclude a potential effect of this
factor on the epigenome of reprogrammed cells that would be
manifested at the time these cells are coaxed to differentiate. A
comprehensive analysis of the epigenetic profiles of OKS-iPSCs vs
OKSM-iPSCs could shed light on the mechanisms behind these
Epigenetic modification of the Gtl2 locus during reprogramming
seems to be important for the generation of iPSCs with full
developmental potential . Our results indicate variability in
Figure 4. Directed differentiation of transgene-free iPSCs into hepatic progenitors is not affected by the epigenetic status of the
Dlk1-Dio3 gene cluster. qRT-PCR analysis of the differentiating cultures at different time points demonstrates induction of the endoderm marker
Sox17 followed by an increase in expression of the liver markers AFP and albumin. Remarkably, all iPSC clones displayed an in vitro differentiation
capacity comparable to ESCs regardless of the expression levels of Gtl2 and Dlk1.
Gene Dysregulation in Transgene-Carrying iPSCs
PLOS ONE | www.plosone.org7 December 2012 | Volume 7 | Issue 12 | e51711
the frequencies of mis-imprinted iPSC clones derived by
reprogramming dermal fibroblasts. Importantly, all iPSC lines
were derived from male mice, thus ruling out the possibility that
the epigenetic differences might be due to the lower global and
DMR-specific methylation previously reported for female ESCs
. Specifically, we found that reprogramming of C57BL/6
fibroblasts by means of a single polycistronic vector gave rise to
hypermethylated Gtl2OFFiPSC clones, while the same experi-
mental approach yielded mostly clones with low to normal Gtl2
methylation levels when C57BL/66129/sv fibroblasts were trans-
duced. Moreover, two clones underwent Gtl2 silencing after
transgene excision, suggesting that the reprogramming factors may
be directly involved in this process. In support of this idea, binding
sites for Oct4 and Klf4 were identified in the Dlk1-Dio3 region, and
Chip-qPCR analysis revealed increased recruitment of Klf4 near
the TSS of Gtl2 in transgene-carrying iPSCs. Our findings imply
an active role of Klf4 (and possibly Oct4) in establishing the
methylation status of Gtl2 and suggest that, when present at
supraphysiological levels, Klf4 may protect this region from
cytosine methylation through a mechanism similar to that
described for the imprinted Igf2 gene . These results are in
concordance with recent studies emphasizing the role of Klf4 in
establishing appropriate Gtl2 imprinting . In addition, some of
the epigenetic modifiers identified as differentially expressed in our
microarray analysis could also play a role. For example, Dnmt3a,
which was found to be downregulated in transgene-carrying
iPSCs, methylates Gtl2/Dlk1 DMRs in vivo . Another possible
alternative is that Oct4 and Klf4 binding promotes the activity or
recruitment of DNA methyltransferases thus altering the methyl-
ation status of the Dlk1-Dio3 locus. A recent study by Stadtfeld
et al. provides experimental support for this last hypothetical
model by demonstrating that hypermethylation of the Dlk1-Dio3
IG-DMR occurs late in the reprogramming process and is
catalyzed by Dnmt3a . Moreover it is suggested that the
absence of ascorbic acid results in loss of the histone acetylation
active marks at the chromatin state thus facilitating the re-
cruitment of Dnmt3a and the resulting hypermethylation of Dlk1-
Dio3 locus . In combination our data and these recent findings
suggest that Oct4 and Klf4 binding at the Dlk1-Dio3 locus results
in chromatin alterations marked by hypomethylation of the locus
when the reprogramming genes are constitutively expressed.
Removal of the reprogramming cassette results in resolution of
the DMRs in this locus to a normal (50% methylated) or
aberrantly imprinted (hypermethylated) state. Indeed, different
combinations/stoichiometries of reprogramming factors appear to
have an effect on the epigenetic status of Gtl2 as recently shown
. Lastly, further studies are needed to address the possibility
that aberrant imprinting is affected by cellular changes that
accompany the subcloning and expansion of iPSCs.
In summary, we demonstrate that residual expression of
exogenous reprogramming factors has a pervasive effect on the
transcriptional program of mouse iPSCs and may also influence
epigenetic signatures associated with full pluripotency. Although
retroviral and lentiviral vectors undergo silencing in human
pluripotent cells, our findings suggest that variegation effects as
well as potential reactivation of the transgenes during differenti-
ation could have a negative impact on the biological properties of
iPSCs. Indeed, even small variations in the levels of pluripotency
factors appear to have a profound effect on the early cell fate
choices of ESCs . Collectively, our data demonstrate the
importance of generating iPSCs that are free of reprogramming
transgenes for both research and therapeutic applications.
Materials and Methods
Generation and Characterization of iPSCs
STEMCCA-loxP and EF1a-STEMCCA-RedLight-loxP lentiviral
vectors was done essentially as described in . Briefly, tail-tip
fibroblasts (TTFs) were isolated from either newborn Sox2-GFP/
M2rtTA mice (mixed genetic background C57BL/66129/sv) or
M2rtTA mice (genetic background C57BL/6) and infected at
passage 3. All mice employed in this study were male. All animal
studies were approved by the Boston University IACUC
committee. iPSC clones were isolated, expanded and character-
ized by immunofluorescence, alkaline phosphatase staining,
teratoma formation and karyotyping as previously described
[13,24]. Southern blot analysis was carried out to select iPSC
clones carrying a single copy of the polycistronic vector and to
confirm Cre-recombinase mediated removal of the transgenes
mouse fibroblastswith the EF1a-
Cell Culture and RNA Isolation
The Sox2-GFP/M2rtTA ESC line  was a kind gift of
Konrad Hochedlinger; C57BL/6 ESCs were obtained from
ATCC (ATCC, American Type Culture Collection); and the
ST5 and ST8 iPSC clones have been described previously .
ESCs and iPSCs were cultured on mitomycin C-treated mouse
embryonic fibroblasts in ESC media (DMEM supplemented with
15% FBS, L-glutamine, penicillin/streptomycin, nonessential
amino acids, b-mercaptoethanol and 1,000 U mL21leukemia
inhibitory factor (LIF; ESGRO; Chemicon; Millipore). Undiffer-
entiated ESC/iPSCs were harvested by trypsinization and plated
twice onto cell culture dishes to deplete the feeder cells before
RNA isolation. Total RNA was isolated with the miRNeasy Mini
Kit (Qiagen) and treated with RNase-Free DNase I (Qiagen)
before performing gene expression analysis.
One microgram of RNA was reverse-transcribed using the
TaqMan Reverse Transcription Reagents kit (Applied Biosystems)
according to the manufacturer’s instructions. The STEMCCA and
STEMCCA-RedLight polycistronic viral transcripts were ampli-
fied in a StepOnePlus real-time PCR system (Applied Biosystems)
using Custom TaqManH Expression Assays as described by the
manufacturer. Endoderm induction and liver specification from
iPSCs were evaluated using the following Taqman inventoried
primers andprobes (Applied
(Mm00488363_m1), AFP (Mm00431715_m1)
(Mm00802090_m1). qRT-PCR analysis of Gtl2, Rian and Dlk1
expression was carried out with Power SYBR Green Master Mix
(Applied Biosystems). Primer sequences are described in .
Reactions were performed in duplicate using 1/20 diluted cDNA.
Transcript expression levels were normalized to b-actin, 18S
rRNA, or GAPDH, and relative quantification of expression was
estimated using the comparative Ct method. qRT-PCR analysis of
total and endogenous levels of reprogramming factors was
performed as described previously .
Gene Expression Profiling
The Mouse Gene 1.0 ST array (Affymetrix) was used for
mRNA expression profiling following the manufacturer’s protocol.
Twenty-five raw data files obtained by the Affymetrix scanner
passed data quality control steps prior to RMA (robust multiarray
average) normalization using the Affymetrix Expression Console
software. To determine the differentially expressed genes affected
by either cell type (ESC/iPSC) or transgene excision (before Cre/
Gene Dysregulation in Transgene-Carrying iPSCs
PLOS ONE | www.plosone.org8December 2012 | Volume 7 | Issue 12 | e51711
after Cre), a two-way ANOVA method was applied to analyze all
25 samples comprised of 5 groups representing the differing cell
types (first ANOVA variable) and the effect of transgene excision
(second ANOVA variable). The differentially expressed genes were
subsequently subjected to the multiple hypothesis test by using
FDR adjustment. We have developed similar ANOVA methods
for microarray analyses of iPSCs detailed previously . The
DAVID gene functional classification tool (http://david.abcc.
ncifcrf.gov) was used to identify Gene Ontology (GO) terms and
KEGG pathways that were enriched in the list of differentially
Bisulfite Modification and Sequencing
Genomic DNA was purified with the DNeasy Blood & Tissue
Kit (Qiagen) and modified with sodium bisulfite using the EpiTect
Bisulfite Kit (Qiagen) according to the manufacturer’s instructions.
Pyrosequencing reactions were conducted by EpigenDx using the
ADS935 assay for Gtl2 IG-DMR and the ADS1341 assay for Gtl2
Chromatin Immunoprecipitation (ChIP)
ChIP was performed as previously described [50,51]. Briefly,
chromatin solution was pre-cleared with salmon sperm DNA/
protein A-agarose 50% gel slurry (cat. #16–157; Millipore) for
45 min at 4uC and immunoprecipitated overnight at 4uC. DNA-
histone complex was collected with 60 mL of salmon sperm DNA/
protein A-agarose beads for 1 hr. The beads were sequentially
washed once with low salt (0.1%SDS, 1%Triton, 2 mM EDTA,
20 mM Tris, pH 8.1, 15 mM NaCl), high salt (0.1%SDS,
1%Triton, 2 mM EDTA, 20 mM Tris, pH 8.1, 500 mM NaCl)
and LiCl (0.25 M LiCl, 1% NP-40, 1% Deoxycholic acid, 1 mM
EDTA, 10 mM Tris, pH 8.1) and washed twice with 10 mM Tris
(pH 8)/1 mM EDTA buffers. The DNA-histone complex was
then eluted from the beads with 250 ml of elution buffer (1% SDS,
0.1 M NaHCo3). DNA and histones were reverse crosslinked at
65uC for 4 hr under high-salt conditions. Proteins were digested
using proteinase K treatment for 1 hr at 45uC. The DNA,
associated with methylated histones, was extracted with phenol/
chlorophorm/isoamyl alcohol, precipitated with 70% ethanol, and
finally resuspended in 80 mL of DDW. Real-time PCR (CFX96,
BioRad) reactions were performed in triplicates and each
experiment was repeated 2–3 times. This experiment was done
using all iPSC/ESC lines except for clones ESC 1, OKSM F,
OKSM-Cre F, OKS 5 and OKS-Cre 5.
In vitro Differentiation Assay
All ESC/iPSC lines were adapted to serum-free maintenance
media  prior to differentiation. Cells were induced to form
definitive endoderm followed by hepatic specification in serum-
free differentiation medium (SFD) as previously described . In
brief, cells were aggregated in 100-mm bacteriological dishes
(Fisher Scientific) and allowed to form embryoid bodies (EBs) for 2
days in the absence of LIF followed by trypsinization and
reaggregation for 2 more days in SFD containing 50 ng/ml
Activin A (R&D Systems). At day 4 EBs were trypsinized and
reaggregated in SFD medium supplemented with 200 mM L-
glutamine, 4.561024M MTG, 50 ng/ml Activin A, 50 ng/ml
BMP-4, 10 ng/ml bFGF, and 10 ng/ml VEGF (all from R&D
Systems). At day 5, EBs were trypsinized, plated on gelatin-coated
plates and cultured in the same medium without Activin A but
supplemented with 20 ng/ml EGF, 20 ng/ml HGF, 20 ng/ml
TGF-a (all from R&D Systems), and 1027M dexamethasone.
STEMCCA vectors. (A) Southern blot analysis was performed
to select iPSC clones carrying a single copy of the polycistronic
vector that is excised after treatment with Cre-recombinase.
gDNA was digested with BamHI and probed using standard
methods. Clones 5, 15, B and D are shown as an example. Each
band represents a single viral integration that is not detected after
exposure to Cre-recombinase. (B) iPSCs derived using the
STEMCCA vectors display ESC-like colony morphology (Phase),
Sox2-GFP reporter gene expression, and alkaline phosphatase
activity (Alk Phos), and form teratomas containing tissues derived
from all three germ layers after injection into immunocompro-
mised mice. (C) Representative images of DAPI-stained metaphase
chromosomes from actively growing iPSCs displaying a normal
karyotype (2n=40) both before and after transgene excision.
Characterization of iPSCs generated with the
unique differentially expressed probesets between the
different iPSC ‘‘cell types’’ and the ESC control group
(two-way ANOVA; p, ,0.0001) (top) or between iPSCs
generated with 3 and 4 factors (bottom). Tables show the
numbers of differentially expressed probesets according to in-
creasing p values.
Venn diagrams illustrating common and
nous levels of the reprogramming factors in the 25
samples profiled by microarray.
RT-qPCR analysis shows total and endoge-
and post-Cre iPSCs (FDR-adjusted p-value , ,0.01).
Differentially expressed probesets in pre-Cre
Gene ontology analysis of differentially ex-
Log2values of genes associated with enriched
score , ,0.05).
KEGG pathways identified by DAVID (EASE
Log2values of genes that map to the TGF-beta
Summary of the iPSC/ESC lines used in this
Conceived and designed the experiments: AS EM GM DNK. Performed
the experiments: CC AGS BSS HH. Analyzed the data: CAS CC AGS
SSS AS EM GM DNK. Wrote the paper: CAS GM DNK.
Gene Dysregulation in Transgene-Carrying iPSCs
PLOS ONE | www.plosone.org9December 2012 | Volume 7 | Issue 12 | e51711
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Gene Dysregulation in Transgene-Carrying iPSCs
PLOS ONE | www.plosone.org10 December 2012 | Volume 7 | Issue 12 | e51711