EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Opposing Putative Roles for Canonical and Noncanonical NFjB
Signaling on the Survival, Proliferation, and Differentiation
Potential of Human Embryonic Stem Cells
CHUNBO YANG,aSTUART P. ATKINSON,a,bFELIPE VILELLA,bMARIA LLORET,bLYLE ARMSTRONG,a,b
DEREK A. MANN,cMAJLINDA LAKOa,b
aInstitute of Human Genetics, International Centre for Life, Newcastle University, Newcastle Upon Tyne, United
Kingdom;bCentro de Investigacio ´n Prı ´ncipe Felipe, Valencia, Spain;cLiver Research Group, Institute of Cellular
Medicine, Newcastle University, United Kingdom
Key Words. Human embryonic stem cells•Canonical and noncanonical NFjB signaling•Pluripotency•p65•RELB•CYCLIN D1
The canonical and noncanonical NFjB signaling pathways
regulate a variety of cellular activities; however, their func-
tions in human embryonic stem cells (hESCs) have not been
fully investigated. Expression studies during hESC differen-
tiation indicated a significant increase in the expression of
two key components of the canonical NFjB pathway (p50
and Ser529 phosphorylated form of p65) as well as a signifi-
cant reduction in expression of key components of the nonca-
nonical NFjB pathway [v-rel reticuloendotheliosis viral
oncogene homolog B (RELB), p52, NIK]. Inhibition of ca-
nonical NFjB resulted in hESC apoptosis, changes in cell
cycle distribution, and reduced hESC proliferation. In addi-
tion, inhibition of canonical NFjB was associated with signif-
icant changes in NANOG and OCT4 expression, suppression
of differentiation toward all primitive extraembryonic and
embryonic lineages with the exception of primitive ectoderm
and ectodermal lineages. Inhibition of noncanonical NFjB
via small interfering RNA-mediated downregulation of
RELB resulted in reduced hESC proliferation and opposite
changes to expression of key differentiation lineage markers
genes when compared with downregulation of canonical NF-
jB. Chromatin immunoprecipitation assays indicated bind-
ing of p65 and RELB to regulatory regions of key differen-
tiation marker genes suggesting a direct transcriptional role
for both branches of this pathway in hESC. These findings
coupled with opposing trends in expression of key compo-
nents during hESC differentiation, suggests a fine and oppos-
ing balance between the two branches of NFjB signaling
pathways and their involvement in two distinct processes:
the canonical pathway regulating hESC differentiation and
the noncanonical pathway maintaining hESC pluripotency.
STEM CELLS 2010;28:1970–1980
Disclosure of potential conflicts of interest is found at the end of this article.
NFjB was identified 24 years ago as a nuclear factor that
binds the j light-chain enhancer in B cells is a cardinal regu-
lator of inflammatory and innate immune responses . A
large body of research suggests that NFjB activation is impli-
cated in various aspects of cell proliferation, migration, cell
cycle progression, and apoptosis [2, 3]. Activation of NFjB
and its ability to confer cell survival is linked to several types
of cancer including hematological and epithelial malignancies
. On the other hand, inhibition of NFjB might increase the
incidence of cancers of the liver and skin suggesting cell-spe-
cific effects [5–7].
The mammalian NFjB family is composed of five subu-
nits, namely p65 (RelA), v-rel reticuloendotheliosis viral
oncogene homolog B (RelB), c-Rel, p52, and p50,  which
combine to generate a variety of heterodimeric and homodi-
meric transcription factors. The five subunits share a Rel
homology domain, which is responsible for DNA binding,
dimerization, and nuclear translocation. The p65, RelB, c-Rel
proteins are synthesized as mature forms and contain a trans-
activation domain at their C-terminus enabling them to
directly stimulate transcription. The p50 and p52 subunits are
Author contributions: C.Y.: collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of
manuscript; S.P.A.: collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of
manuscript; F.V.: collection and assembly of data and final approval of manuscript; M. Lloret: collection and assembly of data and final
approval of manuscript; L.A.: conception and design, financial support, data analysis and interpretation, manuscript writing and final
approval of manuscript; D.A.M.: conception and design, financial support, data analysis and interpretation, manuscript writing and
final approval of manuscript; M. Lako: conception and design, collection and assembly of data, data analysis and interpretation,
manuscript writing and final approval of manuscript. C.Y. and S.P.A. contributed equally this article.
Correspondence: Majlinda Lako, M.Sc., Ph.D., Institute of Human Genetics (UK) and CIPF (Spain), Newcastle University, International
Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, United Kingdom. Telephone: 44-191-241-8688; Fax:
44-191-241-8666; e-mail: firstname.lastname@example.orgReceived June 8, 2010; accepted for publication September 11, 2010; first published
online in STEM CELLS EXPRESS September 29, 2010. V
STEM CELLS 2010;28:1970–1980 www.StemCells.com
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.528
generated from precursors, p105 and p100, and lack a transac-
tivation domain and cannot stimulate transcription unless part-
nered with p65, RelB, or c-Rel. Interestingly, the C-terminal
region of p105 and p100 contains a series of ankyrin repeats
that are characteristic of inhibitors of NFjB (IjB) [9, 10].
In resting cells, the majority of NFjB subunits are associ-
ated with a family of proteins called IjB, which includes
IjBa, IjBb, IjBe, IjBc, IjBf, BCL3, p100, and p105 .
These proteins act as chaperones of the NFjB subunits and
prevent their binding to DNA. On receiving relevant cell
stimulation, the IjBs are degraded and NFjB is released to
translocate to the nucleus where it binds to jB DNA binding
motifs in the regulatory region of genes controlling inflamma-
tion and cell proliferation, differentiation, and fate.
There are currently three known mechanisms of NFjB
activation . The canonical pathway culminates in the deg-
radation of IjBa and is achieved via activation of the Ikap-
paB kinase (IKK) complex. The core IKK complex contains
two kinases, IKK1/a and IKK2/b along with a noncatalytic
subunit called IKKc, more commonly known as NEMO. Acti-
vation of the IKK complex leads to IKKb-mediated serine
phosphorylation of IjBa triggering polyubiquitination and
proteasome-mediated degradation. In addition to IKK-medi-
ated IjB degradation many other events are essential for ca-
nonical activation, the best characterized so far being phos-
phorylation of p65. This can be achieved by many kinases
such as PI3K/v-akt murine thymoma viral oncogene homolog
(AKT), NFjB inducing kinase (NIK), p38, protein kinase C
(PKC), protein kinase A (PKA), etc . The phosphorylation
of p65 is thought to enhance nuclear transport, increase DNA
binding of NFjB and stabilization of p65 by inhibiting its
binding to IjBa.
The second mode of NFjB activation, the so-called non-
canonical pathway is independent of IKKb and IKKc and is
crucial to lymphoid organogenesis . A small number of
stimuli such as lymphotoxin-b and B-cell activating factor
(BAFF) are known to activate this pathway via an upstream
kinase called NIK. A different IKK complex composed of
IKKa dimers phosphorylates p100, (a sequestrator of RelB),
which is partially processed to generate the p52:RelB dimer.
The third pathway of NFjB signaling is activated in response
to DNA damage caused by agents such as UV or doxorubicin
that results in IjB degradation in IKK-independent fashion
. This results in dimerization of free NFjB subunits that
are then mobilized in a similar way to canonical NFjB
A number of recent publications suggest that NFjB sig-
naling plays a role in stem cell proliferation and survival .
A very relevant example is provided by the activation of the
canonical NFjB pathway, which is shown to stimulate prolif-
eration of neural stem cells via upregulation of the bona fide
target gene cyclin D1 . Genetic loss of both p65 and p50
NFjB subunits also results in reduced numbers of neural pro-
genitors and an increased proportions of neurons suggesting
that the effects of NFjB signaling are likely to affect both the
proliferation and differentiation of neural stem cells . An
overexpression of the NFjB components and targets are also
found to be a major characteristic of hematopoietic progenitor
cells found in umbilical cord blood , although the func-
tional significance of this pathway remains to be evaluated in
this cell type. NFjB pathway also plays an important role as
a survival factor for lymphoid progenitor cells and recent
findings have revealed that inhibition of this pathway impairs
the generation of lymphoid cells from adult bone marrow and
fetal liver hematopoietic stem cells  suggesting that the
activation of this pathway is important for the differentiation
of adult hematopoietic stem cells.
Virtually all members of the NFjB pathway are expressed
in embryonic, trophoblast, and uterine cells in a developmen-
tal stage and cell-specific manner . It has been suggested
that the antiapoptotic effect of NFjB in these embryonic
stages is indispensable for proper development during organo-
genesis and is important for resistance toward stress-induced
processes. More recently, it has been shown that murine and
human embryonic stem cells (hESCs) possess a low level of
NFjB activity that increases significantly during the differen-
tiation process [20, 21]. The low NFjB activity in hESC does
not, however, exclude a role for this pathway. Indeed, the
work done in our group suggests that the NFjB pathway
plays a crucial role in the maintenance of viability in hESC
. In accordance with this, it has been shown that karyo-
typically abnormal hESC and embryonic carcinoma cells
express CD30, a member of tumor necrosis receptor subfam-
ily that is responsible for the activation of the canonical
NFjB pathway . In addition, BAFF, which is known to
induce the noncanonical NFjB signaling pathway is used in
defined medium together with other growth factors for pluri-
potent culture of hESC .
In this article, we have investigated the expression and
role of the canonical and noncanonical NFjB pathways in
proliferation, viability, and differentiation capability of hESC.
Our results suggest that canonical NFjB is involved in hESC
differentiation and hESC viability, while noncanonical NFjB
is mainly implicated in maintenance of hESC pluripotency.
Binding of both p65 and RELB to promoters of several line-
age-specific genes indicates that some of these effects are
likely to be mediated at the level of gene transcription.
MATERIALS AND METHODS
Culture and Differentiation of hESC
The hESC lines H9 and H1 (WiCell, Madison,
USA) were routinely passaged and maintained in hESC media on
mitotically inactivated mouse embryonic fibroblast (MEF) feeder
layers and differentiation was achieved by forming embryoid
bodies (EBs) as described by Stojkovic et al. . In 1–2 pas-
sages prior to experiments, hESCs were transferred to Matrigel-
coated plates with feeder-conditioned media as previously
described . In all figures (unless indicated in figure legends),
average data from both cell lines is presented.
Reverse Transcription Polymerase Chain Reaction
This method is described in Supporting Information Annex A.
Cell Cycle Analysis
This method is described in Supporting Information Annex A.
This method is described in Supporting Information Annex A.
Lysates were subjected to electrophoresis on a 10% SDS-poly-
acrylamide (PAGE) gel and transferred to a polyvinylidene diflu-
oride membrane (Hybond-P, Amersham, Buckinghamshire, UK).
Membranes were blocked in Tris-buffered saline with 5% milk
and 0.1% Tween. Blots were probed with primary antibodies
overnight and revealed with horseradish peroxidase-conjugated
secondary antibodies. Primary antibodies used include NFjB
p105/p50 antibody (Abcam, Cambridge, UK; ab32360), NFjB
p100/52 (Santa Cruz sc-7386), NFjB p65 (Santa Cruz sc-372),
RelB Antibody (Cell Signaling #4954), c-Rel (Santa Cruz sc-70),
NIK Antibody (Cell Signaling #4994), IKKa Antibody (Cell Sig-
naling #2682), NFjB p65 (phospho S529) antibody (Abcam
Yang, Atkinson, Vilella et al.
ab10684), Phospho-NFjB p65 (Ser536) Antibody (Cell Signaling
#3031), OCT4 [POU5F1] antibody, clone 7F9.2 from (Millipore,
Watford, UK; # p20263), NANOG antibody (Aviva Systems
Biology, San Diego, California, USA; # P100591_P050), PAX6
antibody (Millipore # P26367), BETA ACTIN antibody (b-Actin
[C4] from Santa Cruz Biotech. (Heidelberg, Germany) # sc-
47778), and GAPDH antibody (# Abcam ab9485). Antibody-anti-
gen complexes were detected using ECL (Amersham Bioscien-
ces) and images were acquired using a luminescent image analy-
ser FUJIFILM and LAS-3000 software (FUJI).
Small Interfering RNAs and Transfection
hESCs were cultured under feeder-free conditions with feeder
conditioned media free of antibiotics for at least 4 days prior to
transfections. Small interfering RNAs (siRNAs) for RelB and p65
were obtained from Invitrogen (Paisley, UK). The siRNA sequen-
ces are shown in Supporting Information Table 2. Transfection
with scrambled control siRNAs with similar GC content to gene-
specific siRNA sequences provided by the same company were
used as a negative control. Transfection of siRNA into hESC was
carried out using the high efficiency nucleofection kit L from
Amaxa (Cologne, Germany) and 80 pmol siRNA (in 2 ml
medium) as outlined in manufacturer’s instructions (program A-
Luciferase Reporter Assay
Plasmid DNA was prepared using Maxiprep kit (Qiagen, Sussex,
UK). The IjBa-Luc promoter reporter gene construct has been
described elsewhere . hESC differentiation was induced with
differentiation medium (80% knockout Dulbecco’s modified
Eagle’s medium, 20% foetal calf serum (FCS), 1X nonessential
amino acids, 1X L-glutamine/penicillin-streptomycin) and remov-
ing ESC from feeder layers. At various differentiation time
points, differentiated hESC and their undifferentiated counterparts
were dissociated to single cells. Equal cell numbers from all sam-
ples were transfected with 1 lg of reporter plasmid DNA and 10
ng of control Renilla plasmid pRL-TK (Promega, Southampton,
UK) using the Amaxa Cell Line Nucleofector Kit L according to
their instructions (program A-023). After 24 hours, cells were
lysed using the lysis buffer provided in the dual luciferase detec-
tion kit (Promega) and following the manufacturer’s instructions.
The firefly and Renilla luciferase activities were measured in turn
using the LARII and Stop Glow solution and the ratio between
the two was calculated.
Alkaline Phosphatase Staining
This method is described in Supporting Information Annex A.
Flow Cytometry Analysis of hESC
This method is described in Supporting Information Annex A.
Measurement of Cell Proliferation Using 5-Ethynyl-
This method is described in Supporting Information Annex A.
IKK2 inhibitor VI (Calbiochem, Nottingham, UK; 401483) at
concentration of 20 lM, domain (NEMO-binding domain [NBD])
binding peptide wild-type (Calbiochem 480025, concentration of
10 lM) or mutated negative control (Calbiochem 480030, con-
centration of 10 lM) were used throughout this study. The hESCs
were plated in Matrigel in the presence of MEF-conditioned me-
dium as described in . Inhibitors were added the following
day and medium was changed every day for the following 6 days
Chromatin Immunoprecipitation Assays
First bioinformatics analysis using Genomatix software was per-
formed on each gene to identify potential NFkB family binding
sites. DNA oligonucleotides were designed with the aim of
amplifying genomic areas with potential binding sites (PBS) and
as negative control areas without binding sites (NPBS) were used
(Supporting Information Table 3). In brief, cells were harvested
at 70%–80% confluence and chromatin immunoprecipitation
(ChIP) was carried out essentially as in Dahl and Collas .
Sonication was optimized to give chromatin fragments of 100–
500 bp in length and DNA from each immunoprecipitation was
purified using the Qiaquick DNA Purification kit (Qiagen, West
Sussex, U.K.) prior to quantitative polymerase chain reaction
(qPCR) analysis. Also included in the experiment was an IgG
antibody control immunoprecipitate to detect any background
which, if present, was subtracted from each immunoprecipitate
within that experiment.
Two tailed pair wise Student’s t test was used to analyze results
obtained from two samples with one time point. The results were
considered significant if p < .05.
Canonical NFjB Pathway Is Stimulated During
To monitor canonical NFjB activity in hESC and during the
differentiation process, we used an IjBa-Luc promoter re-
porter, which is induced by the p65 transactivating subunit of
NF-kappa B . IjBa-Luc  was cotransfected with
Renilla luciferase into undifferentiated and differentiated
hESC at day 10, 20, and 30 of EB-induced differentiation.
Compared with undifferentiated hESCs, differentiated cells
generated elevated IjBa-Luc activity (Supporting Information
Fig. 1), indicating that canonical NFjB activity is stimulated
during hESC differentiation. This result is in accordance with
a recent report with another hESC line, SNUhES3 .
Differential Expression of NFjB Pathway
Components During hESC Differentiation
To investigate the expression of components of the NFjB
system in hESC differentiation, we used the EB-differentia-
tion method and collected cell samples at 10-day intervals till
day 30. Our previous work has shown that during this differ-
entiation time period, hESC lose the expression of key pluri-
potency markers (such as OCT4, NANOG, SOX2, TERT) and
acquire characteristics of differentiated cells derived from the
three germ layers marked by the increased expression of AFP,
PAX6, and KDR [22, 30]. Quantitative reverse transcription
polymerase chain reaction (qRT-PCR) analysis indicated a
significant and continuous increase in p50/p105 and p65
expression during hESC differentiation, although transcripts
encoding these proteins were present in hESC (Fig. 1).
Increased expression of p50/p105 and p65 was further con-
firmed by Western blotting (Fig. 2). No changes in phospho-
rylation of p65 (Ser536) were observed, although a slight
decrease of this phosphorylated form was noticed by flow
cytometry during differentiation of hESC , which may
reflect a difference in the sensitivity of these two techniques.
Of interest is a sudden increase in phosphorylation of p65
(Ser 529) at day 20 of differentiation followed by a decrease
at day 30 (Fig. 2). The observed upregulation of p65 and p50/
p105 expression is consistent with the enhancement of canoni-
cal NFjB activity during hESC differentiation observed with
The Role of NFjB Signalling on hESC
the IjBa-Luc promoter reporter assay reported in Supporting
Information Figure 1.
qRT-PCR analysis also indicated a significant decrease in
expression of inhibitors IjBb and IjBe as well as a time course
specific increase in expression of activators, IKK2 (day 30),
NEMO (day 10), and BCL3 (day 20 and 30), which may con-
tribute to NFjB activity enhancement (Fig. 1). In addition,
IjBf expression was upregulated. IjBf preferentially associates
with the NFjB subunit p50 and suppress NFjB activity , so
its upregulation may serve as negative feedback of NFjB
scription polymerase chain reaction. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as
mean 6 SEM (n ¼ 4). Statistical analysis was performed using Student’s t test where each differentiation time point was compared with hESC. Abbrevia-
tions: c-REL, v-rel reticuloendotheliosis viral oncogene homolog (avian); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IKK, IkappaB kinase;
RELB, v-rel reticuloendotheliosis viral oncogene homolog B; NAK, NF-kappa-B-activating kinase; NIK, NFjB inducing kinase.
Expression analysis of various components of NFjB pathway during human ESC (hESC) differentiation by quantitative real time reverse tran-
Yang, Atkinson, Vilella et al.
signaling. The expression of IjBa did not change significantly
during hESC differentiation and a similar result was obtained
for IKK1 (Fig. 1).
In contrast to the increased expression of canonical path-
way subunits, the expression of RELB, NIK, and p52
decreased significantly during hESC differentiation at both the
mRNA and protein level (Figs. 1, 2). No significant changes
were observed in expression of IKK1 transcript (Fig. 1); how-
ever, a decrease in IKK1 protein expression was suggested
by Western blot (Fig. 2). These findings suggest that nonca-
nonical NFjB is suppressed during hESC differentiation.
There are potentially two mechanisms underlying this process
and these include direct downregulation of expression of
p100/p52, NIK, IKK1, and RELB and inhibition of p100 pro-
teasomal processing through downregulation of NIK and sub-
sequent decrease of IKK1 activity .
Inhibition of Canonical NFjB Signaling Results
in Loss of hESC Viability and Modulation of
We next investigated the function of canonical NFjB signal-
ing in hESC initially employing a selective IKK inhibitor
(Calbiochem IKK inhibitor VI). Inhibition of IKK led to loss
of tight and compact hESC colony morphology compared
with hESC cultures treated with vehicle control, dimethyl
sulfoxide (DMSO) (Fig. 3A). The IKK inhibitor-treated hESC
cultures developed gaps indicative of cell death. Flow cytom-
etry analysis confirmed this idea, with increases in the per-
centage of early (Annexin Vþ/ 7-AAD?) and late (Annexin
Vþ/7-AADþ) apoptotic cells compared with the control group
(Fig. 3B). In addition, suppression of IKK caused a decrease
in hESC proliferation indicated by reduced numbers of cells
in S-phase (Fig. 3C). This was further confirmed by propi-
dium iodide flow cytometry, which showed a higher percent-
age of cells in G2/M phase of the cell cycle (data not shown)
compared with DMSO control group. Increasing evidence
suggests that NFjB plays a pivotal role in regulating apopto-
tic responses by transactivating the expression of antiapoptotic
genes in a variety of cell types including primary B cells [33–
38], hepatocytes [39, 40], and primary rat cortical neurons
[41, 42]. Our results in the hESC system corroborate these
findings and suggest that inhibition of canonical NFjB
reduces hESC viability.
qRT-PCR analysis indicated higher NANOG and OCT4
expression in hESC treated with IKK inhibitor (Fig. 3D), this
is consistent with a recent report in murine ESC, where inhi-
bition of NFjB lead to a detectable increase in Nanog and
Oct4 expression . Alkaline phosphatase staining assays
showed no effect of IKK inhibition on numbers of positively
stained colonies and this was corroborated by the flow cytom-
etry analysis showing no changes in the percentage of cells
staining with cell surface marker SSEA4 (data not shown).
Notwithstanding this, the mean intensity of staining for
SSEA4 was significantly higher in cells treated with the IKK
inhibitor (p ¼ 5.49 E ?05; n ¼ 3; IKK inhibitor VI mean
staining 59,831 6 1,698; DMSO-treated group: 41,562 6
417). qRT-PCR revealed downregulation of expression of
marker (GATA6), mesodermal marker (BRACHYURY), and
endodermal marker (IHH) in hESC treated with IKK inhibitor
(Fig. 3E). We also observed a marked increase in the expres-
sion of FGF5 and PAX6 on inhibition of IKK (Fig. 3E); this
may be suggestive of acquisition of primitive ectoderm or
ectodermal features. We used Western blotting technique to
confirm some of these findings at the protein level. Although
the increase in PAX6 expression was confirmed, a significant
decrease in protein level for both OCT4 and NANOG was
observed (Supporting Information Fig. 2) suggesting a more
complex regulation at post-transcriptional level for OCT4 and
NANOG on inhibition of canonical NFjB signaling, which
merits further investigation. A decrease in these markers with-
out noticeable changes in expression of SSEA4 and alkaline
phosphatase staining suggest the emergence of different cell
subpopulation with propensity to differentiate into ectodermal
lineages. Similar changes in marker expression to the ones we
have identified have been reported during the 72 hours of
neuralization of hESC  and or their commitment to an
epidermal pathway  and suggest that inhibition of canoni-
cal NFjB signaling could be clinically relevant for directing
differentiation of hESC to ectodermal derivative lineages.
To confirm our results obtained with the IKK inhibitor and
build a stronger case for function of canonical NFjB as a regu-
lator of hESC differentiation, we treated hESC with the canoni-
cal NBD inhibitor (Calbiochem 480025), which blocks IKKb
activation of NF-jB. We again observed loss of compact hESC
colony formation and development of gaps between cells (Sup-
porting Information Fig. 3A) consistent with increased apopto-
sis, which was confirmed by flow cytometry analysis (Support-
ing Information Fig. 3B). These effects were not observed with
a control mutant NBD peptide. RT-PCR analysis indicated an
increase in NANOG, OCT4, FGF5, and PAX6 expression in
NBD-treated hESC, again corroborating data obtained with the
Calbiochem IKK inhibitor (Supporting Information Fig. 3C).
To further verify these data, we specifically perturbed canonical
NFjB by siRNA-mediated knockdown of p65 in hESC (Sup-
porting Information Fig. 3D). Depletion of p65 was associated
with increased cell death (25% of dead cells in p65 siRNA
group compared with 12.5% in control siRNA group, n ¼ 3)
and with a significant increase in FGF5 and PAX6 expression
(Supporting Information Fig. 3D).
way during human ESC differentiation by Western blotting. The
images are representative of at least three independent experiments.
Abbreviations: c-REL, v-rel reticuloendotheliosis viral oncogene
homolog (avian); GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
IKK, IkappaB kinase; RELB, v-rel reticuloendotheliosis viral oncogene
homolog B; NIK, NFjB inducing kinase.
Expression analysis of various components of NFjB path-
The Role of NFjB Signalling on hESC
graph of hESC treated for 48 hours with vehicle alone (DMSO, left-hand side panel) and IKK2 inhibitor VI (right-hand side panel). (B): Schematic
graph showing an increase in the number of early apoptotic cells (Annexin Vþ/ 7 AAD?) and apoptotic cells (Annexin Vþ/7-AADþ) as result of
application of IKK inhibitor. (C): Schematic graph showing a decrease in number of proliferating hESCs as result of application of IKK inhibitor.
(D): Changes in expression of pluripotent markers estimated by quantitative real time reverse transcription polymerase chain reaction (RT-PCR) 48
hours after treatment with IKK inhibitor. The value for the control cells (treated with DMSO only) was set to 1 and all other values were calculated
with respect to that. (E): Changes in expression of lineage markers estimated by quantitative real time RT-PCR 48 hours after treatment with IKK in-
hibitor. The value for the control cells (treated with DMSO only) was set to 1 and all other values were calculated with respect to that. (F): Changes
in expression of lineage markers in embryoid bodies (EBs; day 7) made from hESC treated with IKK inhibitor or DMSO prior to differentiation pro-
cess. Note that neither DMSO nor IKK inhibitor are present in the culture medium during the whole 7 days of differentiation. The value for the con-
trol cells (treated with DMSO only) was set to 1 and all other values were calculated with respect to that. (G): Changes in the expression of lineage
markers in EBs (day 7) made from untreated hESC. Note that DMSO and IKK inhibitor are added for the whole duration of differentiation to the cul-
ture medium. The value for the control cells (treated with DMSO only) was set to 1 and all other values were calculated with respect to that. (B–G):
Data is presented as mean 6 SEM (n ¼ 3). Statistical analysis was performed using Student’s t test. Abbreviations: 7ADD, 7 amino-actinomycin D;
DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-20-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IKK, IkappaB kinase.
Impacts of inhibition of canonical NFjB signaling on human ESC (hESC) viability, proliferation, and gene expression. (A): Microphoto-
To observe whether the effects of suppression of canoni-
cal NFjB signaling are reversible, we treated hESC with IKK
inhibitor for 48 hours and on its removal we induced differen-
tiation using the EB method for 7 days. RT-PCR analysis sug-
gested a suppression of differentiation toward primitive troph-
oectoderm, primitive endoderm, endoderm, and mesoderm as
shown by diminished induction of CDX2, GATA6, IHH, and
BRACHYURY, respectively (Fig. 3F). In contrast, FGF5 and
PAX6 were upregulated in these EBs compared with control
group, suggesting a preferential differentiation toward ecto-
dermal lineages on inhibition of canonical NFjB signaling for
a limited time at hESC stage (Fig. 3F).
To investigate the role of canonical NFjB signaling dur-
ing the early stages of hESC differentiation, we made EBs
and subjected them for 7 days to IKK inhibitor or DMSO.
Although the two groups of EBs appeared morphologically
similar (data not shown) qRT-PCR analysis suggested a lesser
differentiation toward primitive trophoectoderm, primitive
endoderm, endoderm, and mesoderm (Fig. 3G). Again a pref-
erential differentiation to primitive ectoderm and neuroecto-
derm was suggested by a higher upregulation of FGF5 and
PAX6 markers, respectively (Fig. 3G).
In summary, canonical NFjB signaling appears to be
required in the maintenance of viability of hESC and may play
an important role in hESC differentiation. These results could be
explained by a direct effect of p65/p50 binding on the promoters
of the affected genes or by a more general effect of canonical
NFjB signaling on hESC viability, which results in lower cell
numbers and loss of hESC niche characteristics. To investigate
the first hypothesis, we performed ChIP assays using a p65 ChIP
grade antibody. Several positive controls such as c-MYC, IKKa,
and IKKb, shown to be bona fide transcriptional targets of p65
were included in addition to a negative control gene, TIMP1
(Fig. 4A). Statistical analysis of the results using Student’s t test
comparison methods between each gene tested and the negative
control, TIMP1 gene indicated significant binding to the pro-
moters of three positive control genes as expected. Significant
binding was also noticed at the p52/p100 locus (Fig. 4A).
To analyze binding of p65 to key pluripotency and lineage
markers, we first performed bioinformatics analysis to identify
genomic areas with predicted p65 binding sites, which we
named PBS and also control genomic areas within the same
gene but without predictive binding sites, which we named
NPBS. This analysis revealed potential p65 binding sites within
NANOG, SOX2, GATA6, BRACHYURY, CDX2, PAX6, and
CYCLIN D1 (CCND1); however, we were unable to find poten-
tial binding sites within the 50regulatory region of OCT4. ChIP
was performed in hESC and Student’s t test comparison
method was performed between areas defined as PBS and
NPBS (Fig. 4B). Significant binding of p65 was noticed in the
CYCLIN D1 promoter, which may be the causative factor
behind reduced cell proliferation on inhibition of canonical
NFjB signaling. In addition, significant binding was noticed in
the regulatory regions of FGF5, GATA6, CDX2, and PAX6,
which fits well with data presented in Figure 3 and Supporting
Information Figure 3. Together, these finding would suggest a
role for p65 in maintenance of pluripotency and lineage differ-
entiation by direct suppression at transcriptional level for
FGF5 and PAX6, direct activation at transcriptional level for
CDX2 and GATA6 as well as a role in cell proliferation by
direct binding to CYCLIN D1 promoter.
Downregulation of RELB Impacts Proliferation of
hESC and Gene Expression of Lineage Markers
During hESC differentiation, expression of RELB and p52
was significantly and gradually suppressed (Fig. 1), suggesting
that the noncanonical NFjB signaling pathway may play a
critical role in hESC renewal and pluripotency. To investigate
this, we used RNA interference, which resulted in fivefold
reduction in expression of RELB resulting in remaining of
20% of expression compared with control siRNA sample
(Fig. 5A, 5B). This was also confirmed by Western blotting
(data not shown). qRT-PCR analysis indicated that RELB
knockdown did not result in changes in expression of two plu-
ripotentency markers (NANOG and OCT4, Fig. 5B) but
caused a significant downregulation of SOX2. Notwithstand-
ing, downregulation of RELB caused an increase in expression
of mesodermal marker (BRACHYURY), trophoectodermal
marker (CDX2), primitive endodermal marker (GATA6), and a
reduction in expression of primitive ectoderm marker (FGF5)
and ectodermal marker (PAX6). Upregulation of GATA6 was
also confirmed by immunocytochemistry (Supporting Informa-
tion Fig. 4). Compared with the control group, there were no
significant changes in percentages of apoptotic cells (Fig. 5C),
percentage of cells staining with the cell surface marker
SSEA4, or the percentage of pluripotent colonies detected
with alkaline phosphatase staining on knockdown of RELB
(data not shown). A small but significant reduction in num-
bers of proliferating hESC was observed on knockdown of
RELB (Fig. 5D) using EdU incorporation combined with flow
cytometryanalysis. Theseresultswere reproduced by
enrichment of IKKa, IKK, p52/p100, and c-MYC promoter fragments
after chromatin immunoprecipitation with p65 antibody in human
ESC (hESC). The data represent the mean 6 SEM from four experi-
ments carried out in H9 cell line. Student t test was carried out to
assess significant binding above the level observed for the negative
control TIMP1 gene. (B): Bar chart showing enrichment of CYCLIN
D1, FGF5, GATA6, CDX2, and PAX6 promoter fragments after chro-
matin immunoprecipitation with p65 antibody in hESC. The data rep-
resent the mean 6 SEM from four experiments carried out in H9 cell
line. Student t test was carried out to assess significant binding above
the level observed for the region predicted not to contain any p65
binding sites. Abbreviation: ChIP, chromatin immunoprecipitation.
ChIP assays using p65 antibody. (A): Bar chart showing
The Role of NFjB Signalling on hESC
for control siRNA (left-hand side panel) and RELB siRNA (right-hand side panel) at 48 hours post-transfection. (B): Changes in expression of line-
age markers estimated by quantitative real time reverse transcription polymerase chain reaction 48 hours post-transfection of RELB siRNA in hESC.
The value for the control siRNA-treated cells was set to 1 and all other values were calculated with respect to that. (C): Schematic graph showing
no change in the number of apoptotic cells (Annexin Vþ/7-AADþ) as result of RELB knockdown by RNA interference at 48 hours post-transfec-
tion. (D): Schematic graph showing a decrease in the number of proliferating hESCs as result of RELB knockdown by RNA interference at 48 hours
post-transfection. (B-D): Data is presented as mean 6 SEM (n ¼ 3). Statistical analysis was performed using Student’s t test. (E): ChIP assays using
RELB antibody. Bar chart showing enrichment of IKKa, IKKb, and p52/p100 promoter fragments after ChIP with RELB antibody in hESC. The
data represent the mean 6 SEM from four experiments carried out in H9 cell line. Student t test was carried out to assess significant binding above
the level observed for the negative control TIMP1 gene. (F): ChIP assays using RELB antibody. Bar chart showing enrichment of SOX2, CDX2,
BRACHYURY, and PAX6 promoter fragments after ChIP with RELB antibody in hESC. The data represent the mean 6 SEM from four experiments
carried out in H9 cell line. Student t test was carried out to assess significant binding above the level observed for the region predicted not to contain
any RELB binding sites. Abbreviations: 7ADD, 7 amino-actinomycin D; ChIP, chromatin immunoprecipitation; EdU, 5-ethynyl-20-deoxyuridine;
NPBS, negative control areas without binding sites; PBS, potential binding sites; RELB, v-rel reticuloendotheliosis viral oncogene homolog B.
Impacts of RELB inhibition on human ESC (hESC) viability, proliferation, and gene expression. (A): Microphotograph of hESC treated
propidium iodide staining and flow cytometry analysis (data
not shown). Together these data suggest that RELB is likely
to have an impact on hESC proliferation and activation and
repression of key lineage markers.
To investigate whether these effects are mediated by
direct binding of RELB to pluripotency or differentiation
markers, we performed ChIP assays. Several positive controls
including IKKa, IKKb, and p52/p100 shown to be bona fide
transcriptional targets were included in addition to negative
control gene, TIMP1 (Fig. 5E). Statistical analysis of the
results using the Student’s t test comparison methods between
each gene tested and the negative control, indicated significant
binding to the promoters of the three positive control genes as
Significant binding of RELB was noticed in the regulatory
regions of SOX2, BRACHYURY, PAX6, and CDX2 that fits
well with data presented in Figure 5B. Together, these finding
would suggest a role for RELB in maintenance of pluripo-
tency and lineage differentiation by direct suppression at tran-
scriptional level for BRACHYURY and CDX2 and direct acti-
vation at transcriptional level for SOX2 and PAX6.
Because of their feature of unlimited self-renewal and capacity
to differentiate into cells of all the three germ layers, hESC
have been proposed for regenerative medicine and tissue
replacement after injury or disease. In view of this, the identifi-
cation and study of signaling pathways required for hESC pro-
liferation and differentiation is important for understanding
early human embryonic developmental biology and for clinical
cell therapy. The NFjB signaling pathway plays an important
role in inflammatory and immune responses, apoptosis, trans-
formation, cell adhesion, oxidative stress responses, embryo de-
velopment, hematopoiesis, as well as neuronal functions via the
induction of certain growth and transcription factors [45–47].
But the impacts of NFjB signaling pathway on hESC have not
been fully examined and have formed the topic of this article.
In this study, H1 and H9 hESCs were used as a model
system to study the activity of canonical and noncanonical
NFjB signaling. We found that canonical NFjB activity
was stimulated and expression of p65 and p50 were
enhanced during hESC differentiation. Our studies showed
that p65 was able to bind at the regulatory regions of sev-
eral genes that mark the development of primitive ectoderm
(FGF5), ectoderm (PAX6), trophoectoderm, (CDX2) and
primitive endoderm (GATA6). It is of interest to note that
binding of p50 and p65 to the CDX2 promoter has also
been observed in adenocarcinomas and esophageal cancer
cells  and FGF5 has also shown to be induced in human
fibroblasts as result of activation of canonical NFjB signal-
ing . Although no published data exist for direct interac-
tion between canonical NFjB signaling and GATA6 or
PAX6, our results showing transcriptional changes for all
four genes in response to inhibition of this signaling branch
coupled with ChIP assays argue for a direct binding of p65/
p50 complex at the regulatory regions of these genes. This
would, however, need to be further investigated by ChIP
assays with coactivators and corepressors of the pathway
and is currently ongoing in our group. Inhibition of the ca-
nonical NFjB signaling resulted in a significant increase in
transcription of NANOG and OCT4 expression as well as
decreases at the protein level, although our ChIP assays did
not show direct binding of p65 to the regulatory regions of
these genes. We are more inclined to believe that changes
in the expression of these two pluripotency genes reflect
suppression of cell differentiation to several primitive and
definitive lineages (such as differentiation to primitive troph-
oectoderm and endodermal lineages) and appearance of a
more homogenous and ectodermally poised cell population
in culture. Previously published data in the murine ESC sys-
tem has shown that Nanog binds to NFjB proteins, and this
binding inhibits NFjB transcriptional activity, resulting in
maintenance of murine ESC pluripotency . It seems that
in hESC the mechanisms by which NFjB interacts with plu-
ripotency markers such as OCT4 and NANOG are more
complex and need to be explored further.
Despite the molecular mechanisms that may be involved in
regulation of gene transcription by canonical NFjB signaling,
our findings have direct implications for directing differentiation
to particular lineages. Of outermost relevance is enhancement of
ectodermal differentiation on inhibition of canonical NFjB sig-
naling that can be used in defining conditions for neuronal, reti-
nal, as well as epidermal differentiation studies from pluripotent
stem cells. This also has implications for genetic diseases and
cancer-related studies that have revealed an association between
these diseases and NFjB signaling but have failed to identify
NFjB-regulated genes that may underline the disease pheno-
type. For example, mutations at the EDARADD locus completely
abrogate NFjB activation and result in ectodermal dysplasias
[49, 50]. Equally, constitutive activation of NFjB signaling is
often found in lung, liver, pancreatic, and esophageal cancers
, and given the new interactions, we have revealed here
between NFKB and GATA6 trancription factors often known to
be involved in gut and cardiovascular development, new insights
can be gathered toward new cancer treatments. For example,
some Gata factors such as GATA4 and GATA5  have shown
to be epigenetically silent in gastric cancers, thus changing the
balance between cell differentiation and proliferation. In view of
this, it would be interesting to examine the expression of GATA6
in the stem cell compartment of endodermal type cancers that
show activation of canonical NFjB signaling.
Another aspect of our studies is the increased expression
of p105/p50 together with dramatic upregulation of Bcl3 that
is known to act both as coactivator or corepressor through
association with p50 homodimers. Furthermore, Bcl3 expres-
sion can be induced by NFjB and this which forms a part of
the autoregulatory loop that controls the nuclear residence of
p50 NFjB. Future studies should therefore be directed at
determining the role of Bcl3 as a regulator of NFjB activities
in differentiating hESC.
Our observation for a dramatic decrease in expression of
the noncanonical factors RELB, p52, and NIK during hESC
differentiation raises the possibility that this pathway way may
be a key determinant for the switch between hESC prolifera-
tion and differentiation. It is indeed noteworthy that BAFF, a
known activator of noncanonical NFjB is included in the
media used to maintain pluripotent hESC cultures. We were
able to demonstrate binding of RelB to lineage-specific marker
genes and knockdown of RelB was associated with increased
expression of these differentiation markers supporting our pro-
posal that noncanonical NFjB may function as a suppressor of
hESC differentiation. Of note, the p52 component of nonca-
nonical NFjB has previously been shown to either stimulate or
repress cell proliferation depending on the cell type studied ,
it will therefore be instructive to determine those cell cycle
genes that are targets for p52 in pluripotent hESC. An interest-
ing observation concerning the diminished expression of RelB
and p52 with hESC differentiation was the more dramatic loss
at protein versus transcript level. This may be indicative of
post-transcriptional control mechanisms either at the level of
increased rates of protein degradation or possibly translational
The Role of NFjB Signalling on hESC
control via a micro RNA-related mechanism. Given our limited
understanding of the control of NFjB subunit expression, the
hESC differentiation process provides an interesting model sys-
tem to study mechanisms of regulation of subunit expression.
In summary, we conclude that canonical and noncanonical
NFjB signaling is multifunctional in the control of hESC
fate, and moreover, that hESC differentiation is accompanied
by a switch from noncanonical to canonical NFjB signaling
that may be critical for survival and differentiation in the
postpluripotent state. Further investigations into the specific
functions of the NFjB subunits and their coregulators should
improve our understanding of the transcriptional regulation of
hESC pluripotency, differentiation, and survival.
We are grateful to Ian Dimmick and Dr. Rebecca Stewart for
help with the flow cytometry analysis, Dennis Kirk for technical
assistance and Mann’s lab for providing most of the reagents
needed for this work. This study was supported by One North
East Regional Developmental Agency, and funds for research in
the field of Regenerative Medicine through the collaboration
agreement from the Conselleria de Sanidad (Generalitat
Valenciana) and the Instituto de Salud Carlos III (Ministry of
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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The Role of NFjB Signalling on hESC