RESEARCH
RESOURCE
Expression Profiling of Nuclear Receptors in Human
and Mouse Embryonic Stem Cells
Chang-Qing Xie,* Yangsik Jeong,* Mingui Fu, Angie L. Bookout,
Minerva T. Garcia-Barrio, Tingwan Sun, Bong-hyun Kim, Yang Xie, Sierra Root,
Jifeng Zhang, Ren-He Xu, Y. Eugene Chen, and David J. Mangelsdorf
Cardiovascular Center (C.-Q.X., J.Z., Y.E.C.), Department of Internal Medicine, University of Michigan Medical Center,
Ann Arbor, Michigan 48109; Departments of Pharmacology and Howard Hughes Medical Institute (Y.J., A.L.B., T.S.,
D.J.M.), Biochemistry (B.-h.K.), and Clinical Sciences (Y.X.), University of Texas Southwestern Medical Center, Dallas,
Texas 75390; Burnett College of Biomedical Sciences (M.F.), University of Central Florida, Orlando, FL 32816;
Cardiovascular Research Institute (M.T.G.-B.), Morehouse School of Medicine, Atlanta, Georgia 30310; University of
Connecticut Stem Cell Institute and Department of Genetics and Developmental Biology (S.R., R.-H.X.), University of
Connecticut Health Center, Farmington, Connecticut 06030
Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of
the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression
of NRs between the two human cell lines was highly concordant, whereas in contrast, expression
of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that
have been implicated previously as crucial regulators of mouse ESC biology, including ERR␤,
DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct
species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should
provide a unique resource for further exploration of the species-specific roles of NRs in ESC
self-renewal and differentiation. (Molecular Endocrinology 23: 724 –733, 2009)
mbryonic stem cells (ESCs) derived from early embryos
have unique capabilities of self-renewal and pluripotency
(to differentiate into cell lineages of the three germ layers within
certain environments) (1). Thus, ESCs offer an unprecedented
opportunity to study differentiation events in vitro that mimic
events after implantation in both mice and humans. Furthermore, understanding these events has become increasingly important because of the potential use of ESCs in cell-based replacement therapies (2). Despite the keen interest in ESC
biology, the signal transduction and transcriptional regulatory
pathways involved in the maintenance, differentiation, and manipulation of ESCs still is not completely understood.
To date, the mouse has been the mainstay of mammalian
experimental embryology because of its well-defined genetics
and favorable reproductive characteristics. Indeed, much infor-
E
mation about early human development has been generated
from studies in mouse. However, several fundamental differences exist between mouse and human in early development.
For example, human and mouse embryos differ in the timing of
zygotic genome activation; in the formation, structure, and
function of the fetal membranes and placenta; and in the formation of an embryonic disc instead of an egg cylinder (3–5).
For this reason, it has become desirable and necessary to
pursue comparative studies of mouse and human models of
embryogenesis.
One strategy that has been used to characterize mouse and
human ESCs and identify the unique molecular components
that define each has been genome-wide transcriptome profiling
(6 –11). Although these studies have revealed species-specific
patterns of expression and identified important biomarkers that
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/me.2008-0465 Received December 18, 2008. Accepted January 29, 2009.
First Published Online February 5, 2009
* C.-Q.X. and Y.J. contributed equally to this work and should be considered equal first
authors.
Abbreviations: AKP, Alkaline phosphatase; Ct, cycle time; ESC, embryonic stem cell; FBS,
fetal bovine serum; KO-SR, knockout serum replacement; NR, nuclear receptor; QPCR,
quantitative real-time PCR; SSEA-4, stage-specific embryonic antigen 4; TRA-1-60, tumor
rejection antigen-1-60.
724
mend.endojournals.org
Mol Endocrinol, May 2009, 23(5):724 –733
Mol Endocrinol, May 2009, 23(5):724 –733
define undifferentiated and differentiated states, gleaning further mechanistic insight into how these processes are regulated
has been difficult because, by nature, the genome-wide approach results in datasets that are subjective to open-ended
bioinformatic analyses.
Nuclear receptors (NRs) represent a superfamily of liganddependent transcription factors that govern diverse aspects of
development, reproduction, basal metabolic function, and nutrient uptake and metabolism through a common mechanism of
action (12–14). Included in this superfamily are the classic endocrine receptors that mediate the actions of steroid hormones,
thyroid hormones, and the fat-soluble vitamins A and D; the
lipid-sensing receptors that respond to dietary metabolites of
cholesterol, fatty acids, and xenobiotics; and a number of orphan receptors whose ligands and physiological functions are
still being characterized. Given the importance of NRs in controlling cell differentiation and development, comparing the expression of NRs during early ESC differentiation would be expected to provide a unique view of the early transcriptional
regulatory networks involved in ESC self-renewal and differentiation. With the exception of one report evaluating the expression of receptors for estrogen, progesterone, and glucocorticoids
(15), virtually nothing is known about systemic NR expression
in human ESCs. Therefore, as a first step toward exploring the
species-specific roles of NRs in mouse and human ESC differentiation, in the present study, we characterized the mRNA
expression profile of the NR superfamily in two well-studied,
human ESC lines (H1 and H9) and the mouse ESC line (CMTI-1)
using a high-throughput quantitative real-time PCR (QPCR)
method. Bioinformatic analysis of the resulting expression profiles revealed significant differences in the patterns of expression
of mouse and human NRs in undifferentiated ESCs, underscoring the importance of species-specific studies in stem cell populations. Furthermore, NR profiling during the early differentiation of mouse and human ESCs into embryoid bodies revealed
the existence of a complex, temporally regulated transcriptional
network involving numerous, previously unsuspected nuclear
receptors during early embryonic differentiation. Taken together, our data provide the first comprehensive study of a single
family of transcriptional regulators in ESCs. This information
should serve as a useful resource for exploring species-specific
processes of ESC self-renewal and differentiation.
Results
Characterization of ESCs
Traditional methods to induce ESC differentiation involve
first culturing ESC clumps in suspension for 5– 6 d, after which
they spontaneously form three-dimensional embryoid bodies
(16, 17). Typically, the sphere-like embryoid bodies are then
treated with different stimuli, including growth factors, chemical agents, and matrix factors to drive differentiation into specific cell types. Thus far, different specific cell types derived from
ESCs have been broadly reported (18). However, the low differentiation efficiency and complexity of the resultant cell lineages have made it difficult to illuminate molecular events lead-
mend.endojournals.org
725
ing to cell type-specific differentiation. For this reason, we
concentrated on characterizing the NR expression profiles in
two human (H1 and H9) and one mouse (CMTI-1) ESC line, as
well as during the first 5 (for mouse) or 6 (for human) days of
spontaneous differentiation of these cells as outlined in Fig. 1A.
These cell lines were chosen because of their widespread use in
the stem cell field (e.g. CMTI-1 is the most widely used ESC for
FIG. 1. Experimental design for expression profiling of NRs in ESCs. A,
Schematic representation of ESC differentiation timeline. Undifferentiated ESC
lines were expanded routinely and permitted to differentiate spontaneously into
embryoid bodies with a minimum of 50 cell clumps for human H1 and H9 cells
and a minimum of 106 ESCs for mouse CMTI-1 cells. Differentiated samples
collected every day contained 50 –100 embryoid bodies in both cases. B, Brightfield microscopy showing morphology of undifferentiated hESCs (top left). Cell
colonies exhibit high AKP activity (bottom left) indicated by the intense staining
pattern. Right panels represent immunofluorescence staining of human ESC
colonies with anti-SSEA-4 (top middle and top right) and anti-TRA-1-60 (bottom
right), respectively. As anticipated, ESCs exhibited strong immunoreactivity to
these two antibodies. C, Oct-4, Nanog, AFP, Nestin, GATA-2, and Keratin
expression quantitated by QPCR during spontaneous human (H1 and H9) and
mouse (CMTI-1) ESC differentiation into embryoid bodies. 18S RNA was used as
internal standard. The x- and y-axes represent time (day) after induced differentiation
and relative expression of NRs, respectively. Relative expression levels were
determined by the PCR efficiency-corrected method as described in Materials and
Methods. Values are expressed as mean ⫾ SD from triplicates of each sample.
726
Xie et al.
Nuclear Receptors in ESCs
Mol Endocrinol, May 2009, 23(5):724 –733
generating germline mouse knockouts). The undifferentiated
state of human ESCs was confirmed by immunofluorescence
staining for stage-specific embryonic antigen 4 (SSEA-4) and
tumor rejection antigen-1-60 (TRA-1-60), and the presence of
alkaline phosphatase (AKP) activity (Fig. 1B). Similar analysis
was used to confirm the undifferentiated state of mouse ESCs
(data not shown). In addition, decreased expression of pluripotent gene markers, Oct-4 and Nanog, were monitored by QPCR
in both species of ESCs (Fig. 1C), indicating the progression of
differentiation was occurring as has been observed by others
(19, 20). Further confirmation of the differentiated state was
achieved from observing the appearance of early germ layerspecific gene expression for AFP (an endoderm marker), Nestin
(a neuroectoderm marker), GATA-2 (a mesoderm and trophoblast marker), and keratin (an epidermal ectoderm marker).
Undifferentiated ESCs exhibit species-specific expression
of a core set of NRs
Using a QPCR method developed specifically for NR expression profiling (12, 21), we analyzed the expression of all 49
mouse and 48 human NRs in undifferentiated ESCs and during
their early spontaneous differentiation into embryoid bodies.
The raw and annotated data sets are available as part of the
Nuclear Receptor Signaling Atlas (NURSA), an online open access
resource for nuclear receptor research (www.NURSA.com).
Names and abbreviations of NRs are listed in supplemental
Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.
org); the human and mouse QPCR primer sequences used in this
study are described (supplemental Table 2) (21) and are also
available on the NURSA web site.
Analysis of the mRNA expression profiles revealed that although there was 100% identity in NR expression between the
two human undifferentiated cell lines, only 29 NRs (59%) were
coexpressed in both human and mouse ESC lines (Fig. 2A).
These NRs included AR, COUP-TF␣, COUP-TF␤, COUP-TF␥,
DAX-1, ERR␣, GCNF, GR, HNF4␣, LRH-1, LXR␣, LXR␤,
MR, NGFI-B, NOR1, NURR1, PPAR␦, RAR␣, RAR␤, RAR␥,
REV-ERB␣, REV-ERB␤, ROR␣, RXR␣, RXR␤, TR2, TR4,
TR␣, and VDR. These receptors appear to define a common set
of NRs that may play key roles in ESC self-renewal and differentiation. Surprisingly, five of the NRs (HNF4␥, PPAR␥, PXR,
ROR␤, and TR␤) were expressed only in human ESCs, and eight
(ERR␤, ERR␥, PNR, PPAR␣, PR, ROR␥, RXR␥, and SHP) were
expressed only in mouse ESCs (Fig. 2A). Seven (10%) of the NRs
(CAR, ER␣, ER␤, FXR, PPAR␥2, SF1, and TLX) were undetectable [cycle time (Ct) ⱖ33] in any of the three ESC lines.
Although the profiling results demonstrated that an essential
fraction of NR mRNAs were present in both human and mouse
ESCs, there were significant differences in the relative levels of
these mRNAs when their expression was compared between the
two species (supplemental Table 3). Interestingly, in the two
human cells, REV-ERB␤ and TR4 mRNA expression was more
than 10-fold higher than any of the other NRs (see Figs. 3 and 4
for comparisons of relative expression levels). In contrast, in the
mouse ESCs, RAR␥, NGFI-B, and LXR␤ were the most abundantly expressed, and again at levels that were more than 10-
FIG. 2. Expression of NRs in undifferentiated human and mouse ESCs. A, The
Venn diagram depicts mRNA expression (Ct ⱕ33) of the NR superfamily in
human (H1 and H9) and mouse (CMTI-1) ESCs. B–D, Correlation of the relative
expression levels for 29 NRs that were commonly expressed in undifferentiated
human and mouse ESC lines. Relative expression values between each cell line
are compared by scatter plot. Linear regression analysis was used to obtain the
overall correlation (R2) of NR expression for each cell line pair (H1 vs. H9 in B H1
vs. CMTI-1 in C and H9 vs. CMTI-1 in D). Both x- and y-axes represent normalized
expression levels for each sample. Insets represent magnified scattered plots of
red boxed regions. Note that expression of NR1H5 (also known as FXR␤), which
is a mouse-specific NR and a pseudo-gene in humans, was undetectable in
undifferentiated and differentiated ESCs and therefore was excluded from
further analyses.
fold higher than other NRs (Figs. 3 and 4). Linear regression
analysis based on the relative levels of the core set of NRs
expressed in the undifferentiated cell lines revealed that although the two human ESCs (H1 and H9) showed a nearly
identical pattern of expression (R2 ⫽ 0.98) (Fig. 2B), the
correlations of the expression pattern between either of the
two human cell lines (H1 and H9) and the mouse cell line
(CMTI-1) were insignificant (R2 ⱕ 0.001) (Fig. 2, C and D).
To exclude the possibility that the observed species differences were due to the dominant effects of the small number of
Mol Endocrinol, May 2009, 23(5):724 –733
mend.endojournals.org
727
Dynamic expression of NRs during ESC
differentiation
ESCs are a useful model for studying early events
in development as the generation of ESC-derived
embryoid bodies recapitulates early embryo development (22, 23). To begin to understand the physiological relevance of NRs during the earliest stages
of ESC differentiation, we profiled their expression
during the first 6 d of human and 5 d of mouse ESC
differentiation into embryoid bodies. The dynamic
patterns of expression observed for each of the NRs
during embryoid body differentiation could be divided broadly and arbitrarily into two distinguishing categories: NRs with expression patterns that
were similar between human and mouse (Fig. 3)
and NRs with expression patterns that were different between human and mouse (Fig. 4). NRs with
similar expression patterns could be further divided
into three groups: NRs (COUP-TF␥, GR, and
RAR␥) with decreasing expression throughout differentiation (Fig. 3A), NRs (HNF4␣, NOR1, and
PPAR␥) with increasing expression throughout differentiation (Fig. 3B), and NRs (GCNF, PPAR␦,
ROR␣, and RXR␣) with essentially unchanged expression (Fig. 3C).
Twenty-one NRs showed expression patterns
that were different between species during early development (Fig. 4, A and B). Expression of NRs that
tended to increase during differentiation of human
but decrease during differentiation of mouse ESCs
included COUP-TF␣, LRH-1, NURR1, PPAR␣,
RAR␣, RAR␤, REV-ERB␤, RXR␥, TR2, and TR4
(Fig. 4A). Likewise, expression of NRs that were
shared during human and mouse ESC differentiation, but whose patterns were not correlated, included COUP-TF␤, DAX-1, ERR␣, LXR␣, LXR␤,
MR, NGFI-B, REV-ERB␣, RXR␤, TR␣, and VDR
(Fig. 4B). Perhaps even more importantly, NRs that
were expressed exclusively in either human and
mouse undifferentiated ESCs (see Fig. 2A) maintained this exclusive expression pattern throughout
embryoid body formation. Thus, CAR, HNF4␥,
PXR, ROR␤, and TR␤ were expressed only in huFIG. 3. NRs with similar patterns of expression in mouse and human ESCs during embryoid
man ESC lines [albeit at low levels (Ct 29 –32)] (Fig.
body formation. A, Expression of NRs that tended to decrease upon ESC differentiation. B,
Expression of NRs that tended to increase upon ESC differentiation. C, Expression of NRs that
4C), whereas ERR␤, ERR␥, PNR, PR, ROR␥, and
remained relatively constant during ESC differentiation. Comparisons are between human H1
SHP were detected only in mouse ESCs (Fig. 4D).
and H9 and mouse CMTI-1 ESC lines. The x- and y-axrs represent time (day) after induced
Perhaps the most conspicuous difference was the
differentiation and relative expression of NRs, respectively. Relative expression levels were
determined by the PCR efficiency-corrected method as described in Materials and Methods.
expression of ERR␤, which was highly expressed
Values are expressed as mean ⫾ SD from triplicates of each sample. Ct higher than 32 is
(Ct 22) in mouse ESCs but undetectable in human
considered below the limit of detection. Ct of the highest expressing value for each NR group is
ESCs. In addition, it is of interest to note that the
indicated inside its corresponding bar.
relative expression levels of a number of commonly
expressed NRs varied substantially (⬎10-fold) behighly expressed NRs in each group, similar results were
tween human and mouse (supplemental Table 3). These NRs
obtained by repeating the analysis without including these
highly expressed NRs (insets in Fig. 2, B–D). Taken together,
included DAX-1 (Ct 31 in human vs. Ct 25 in mouse), LRH-1
these data imply that species-specific differences in NR pro(Ct 29 in human vs. Ct 25 in mouse), NGFI-B (Ct 30 in human
files may contribute to distinct transcriptional programming
vs. Ct 26 in mouse), and RAR␥ (Ct 25 in human vs. Ct 21 in
of mouse vs. human ESCs.
mouse). These data further support the notion that a select, small
728
Xie et al.
Nuclear Receptors in ESCs
Mol Endocrinol, May 2009, 23(5):724 –733
undifferentiated cells, heat-map patterns from selfcomparisons and cell line to cell line comparisons
showed that the two human ESC lines shared highly
concordant expression patterns during the first 6 d
of differentiation into embryoid bodies (Fig. 5,
compare A and B with C). These data suggest that
differentiation of the two human cell lines is progressing through a similar phenotypic pathway.
In marked contrast, there were diametric differences in the correlations between human and mouse
ESCs (Fig. 5, compare D with E and F). Indeed,
inspection of the human vs. mouse ESC differentiation patterns revealed a striking, almost mirrorimage relationship. NRs that exhibited one correlation pattern when compared in the mouse cells
exhibited a notably opposite pattern when compared between the mouse and either of the two human cells. Linear regression analysis of these data
confirmed that the dynamics of NR expression between the two human ESCs during embryoid body
formation were highly correlative (R2 ⫽ 0.83),
whereas between the human and mouse ESCs, this
correlation was insignificant (R2 ⬍ 0.001) (Fig. 5,
G–I). Taken together, these marked differences in
NR expression between species imply that during
early differentiation, human and mouse ESCs may
use distinct transcriptional programs.
Discussion
This study provides the first system-based approach
to understanding the role of the NR superfamily in
the maintenance and differentiation of ESCs. Using
a high-throughput QPCR approach, we found NRs
exhibit a highly dynamic, complex pattern of expression in both undifferentiated ESCs and during
early cell lineage differentiation. Given their wellFIG. 4. NRs with dissimilar patterns of expression in mouse and human ESCs during embryoid
known roles as mediators of hierarchical transcripbody formation. A, NRs with expression patterns that trended in opposite directions during
human and mouse ESC differentiation. B, NRs with no concordant expression pattern between
tional networks in postembryonic tissues (12), this
species. C and D, NRs expressed only in human (C) or only in mouse (D) during ESC
work suggests a previously unanticipated role for
differentiation. N.D., Not detected (Ct ⬎32). The x- and y-axes represent time (day) after induced
many of the NRs at the very earliest stages of develdifferentiation and relative expression of NRs, respectively. Values are expressed as mean ⫾ SD
from triplicates of each sample. Ct of the highest expressing value for each NR group is indicated
opment. Perhaps even more intriguing, we found
inside its corresponding bar.
that although human and mouse ESCs express a
common set of NRs, there were substantial interspecies differences in the relative levels of the maset of NRs may have species-specific functions that are crucial for
jority of NRs found in both species. Moreover, during the proESC biology.
cess of early ESC differentiation into embryoid bodies, the
dynamic patterns of NR expression varied between the two
Bioinformatics analysis reveals species-specific
species in an almost diametric fashion. Although we cannot
differences in NR expression during ESC differentiation
exclude the possibility that the major differences we observed in
To further evaluate the essential features that define NR
NR expression between human and mouse undifferentiated
expression during embryoid body formation, and further delinESCs are due to cell-specific growth rates and/or culture requireeate those NRs that share the most similarity between the two
ments, we note that a study comparing human ESC lines in
species, we performed unsupervised cluster analysis of the
different culture conditions found the profile of gene expression
pair-wise Pearson correlation values for the 33 NRs that were
to be similar in all cases (8). This suggests that the differences
commonly expressed (albeit in unique patterns) throughout
observed here between NR expression in undifferentiated huthe differentiation process. Consistent with their expression in
Mol Endocrinol, May 2009, 23(5):724 –733
mend.endojournals.org
729
LRH-1, RARs and RXRs, and TR2 (24 –26). However, of these NRs, only GCNF and a subset of the
retinoid receptors exhibited similar patterns of expression in undifferentiated ESCs and during early
embryoid body formation in both mouse and human. During mouse embryogenesis, GCNF has
been shown to be essential for the repression of
pluripotency genes such as Oct-4 and Nanog, and
also in the initiation of differentiation (25, 27). Numerous reports have shown that disruption of various
members of the retinoid receptor subfamily (both
RARs and RXRs) markedly impair early mouse development (28 –31), and it is well known that retinoic
acid is an important regulator for ESC differentiation
and early development in both mice and humans (32–
36). Therefore, it is likely a similar role for GCNF and
the retinoid receptors exists in humans.
Given the importance of ERR␤, DAX-1, and
LRH-1 in maintaining mouse ESC pluripotency
(37–39), the finding that these NRs exhibited markedly different patterns of expression in human ESCs
clearly warrants further investigation. ERR␤ is believed to maintain the pluripotent state by interacting with Nanog and Oct-4 in mouse ESCs (38, 40,
41). In addition, both genetic and pharmacological
mouse models have revealed a prominent role for
ERR␤ in trophoblast differentiation and placenta
formation (42– 44). Surprisingly, however, although
we found ERR␤ to be expressed abundantly in
mouse ESCs, it was undetectable in human ESCs
and remained so throughout their early differentiation (Fig. 4D). Thus, our observation that ERR␤ is
exclusively expressed in mouse ESCs might underlie
a fundamental, species-specific difference that governs the formation, structure, and function of early
embryonic and placental development (3–5).
FIG. 4. Continued.
man and mouse ESCs are likely to be species specific rather than
arising from culture conditions.
Although previous studies have highlighted the differences in
gene expression that exist between mouse and human ESCs (8,
10, 11), the interpretation of these studies has been made difficult by the lack of mechanistic insight that can be derived from
global gene array analyses. Nevertheless, one common feature
of all these studies has been the finding that only a small core set
of genes is likely to exist that are shared and important for ESC
biology in both species (11). Reinforcing this notion, in our
study, we found a surprisingly small number of NRs commonly
expressed between species that also have been implicated as
crucial to ESC viability and pluripotency. NRs that have been
reported to be required for mouse ESC maintenance and early
differentiation include COUP-TF␣, ERR␤, DAX-1, GCNF,
DAX-1 is another orphan NR that appears to be
crucial in early mouse embryogenesis (45). It has
been implicated in other profiling and experimental
studies in the maintenance of pluripotency, in part
by also interacting with Nanog (39). Further evidence has shown DAX-1 is controlled by STAT3
and Oct-3/4 to maintain the self-renewal of ESCs (46). Consistent with these findings, DAX-1 expression was notably abundant in undifferentiated mouse ESCs, and gradually declined
during early differentiation (Fig. 4B). However, in marked contrast to mouse, DAX-1 mRNA levels were several orders of
magnitude lower in human ESCs, suggesting that its role in
human ESCs may be quite different (Fig. 4B and supplemental
Table 3). Likewise, LRH-1, which is believed to be a key factor
in the maintenance of Oct-4 expression and stem cell proliferation (37, 47), was expressed at relatively high levels in undifferentiated mouse ESCs but was nearly undetectable in differentiated embryoid bodies (Fig. 4A). Again, a diametric pattern of
expression was observed in human ESCs, where LRH-1 mRNA
was expressed at relatively low levels in undifferentiated cells,
730
Xie et al.
Nuclear Receptors in ESCs
FIG. 4. Continued.
and increased during differentiation (Fig. 4A and supplemental
Table 3).
The COUP-TFs are another subfamily of orphan NRs that
have been shown to have important roles in governing neurogenesis and organogenesis in mice (48, 49). Overexpression of
COUP-TF␣ in mouse ESCs can reduce retinoic acid-associated
growth arrest and increase extraembryonic endoderm gene expression, suggesting that COUP-TF␣ modulates the earliest
stages of retinoic acid-mediated embryonic development (26). In
the present study, the mRNA expression patterns of mouse
COUP-TF␣ and ␤ were expressed at relatively low levels during
differentiation, whereas their human orthologs showed striking
dynamic increases in expression during differentiation. Taken
together, these studies call into question the function of these
Mol Endocrinol, May 2009, 23(5):724 –733
prominent factors in human ESC biology and highlight the need for further analysis.
The results from the comparison of mouse and
human NR expression during ESC differentiation
further strengthen the notion that the overall regulatory blueprint is more species dependent than previously appreciated. One interpretation of these
data is that, as observed in the undifferentiated
cells, only a fraction of NRs forms the principle
component set that is needed to drive a common
transcriptional programming during differentiation. However, if this were the case, one would
not have expected that the overall correlation
would be so diametrically different. Another
plausible explanation is that the environmental
niche occupied by each ESC population contributes substantially and in a species-specific way to
the differentiation process. For this reason, we
cannot rule out the important concern inherent
with these studies that spontaneous differentiation to more than one cell type may have occurred
between the different species-specific cell lines,
which could clearly affect which NR is expressed
temporally.
At present, the functional consequences of the
expression for the majority of the human NRs
that are differentially expressed during embryoid
body formation are unknown, and it is not clear
that they are crucial for ESC biology or speciesspecific functions. This notion is supported by the
finding that the germline knockouts of many of
these receptors have no embryonic phenotype.
Nevertheless, the observation that two thirds of
the NRs in this category displayed different expression patterns between species supports the
possibility that innate species specificity is encoded in part by NR expression.
In summary, our results show that NRs may be
involved in self-renewal and maintenance of undifferentiated ESCs and early cell lineage differentiation as reflected by their complex patterns of
variation during this process. At the same time,
we found that major differences may exist in NR
expression between mouse and human ESC populations and
during early differentiation, further stressing the need to account for species-specific differences in this field of research.
Future work may be directed toward understanding the exact
function of each NR in maintaining ESCs in undifferentiated
status and during differentiation with the final goal of generating lineage-restricted progenitor and mature cells suitable
for therapeutic applications. Thus, given their importance as
transcriptional sensors for endocrine hormones and other
lipophilic signaling molecules that govern broad aspects of
developmental, metabolic, and immune response programs,
the characterization of the NR superfamily in undifferentiated and early differentiated ESCs should provide a useful
resource for both basic and clinical studies.
Mol Endocrinol, May 2009, 23(5):724 –733
mend.endojournals.org
731
FIG. 5. Bioinformatic analysis of the NR superfamily expression profile in human vs. mouse ESCs. A–F, Heat maps representing cell line self-comparisons (H1 in A, H9
in B, CMTI-1 in D) or cell line to cell line comparisons (H1 with H9 in C, H1 with CMTI-1 in E, H9 with CMTI-1 in F) of NR expression during ESC embryoid body
formation. The heat maps show unsupervised cluster analysis for pair-wise Pearson correlation values of the 33 NRs that were commonly expressed during
differentiation in all three cell lines. Samples were compared pair-wise for each day of differentiation (d 0 through d 5 for CMTI-1 and through d 6 for H1 and H9).
Increased color brightness indicates NR pairs whose expression was more positively (red) or negatively (green) correlated between samples. G–I, Correlation of the
relative expression levels for 33 NRs that were commonly expressed during differentiation of human and mouse ESCs into embryoid bodies (d 0 through d 5). Relative
expression values between each cell line are compared by scatter plot. Linear regression analysis was used to obtain the overall correlation (R2) of NR expression for
each cell line pair (H1 vs. H9 in G, H1 vs. CMTI-1 in H, and H9 vs. CMTI-1 in I). Both x- and y-axes represent normalized expression levels for each sample. Insets
represent magnified scattered plots of red boxed regions.
Materials and Methods
ESC culture and differentiation
A schematic of the in vitro differentiation procedure for ESCs are
shown in Fig. 1A. In brief, human ESCs (H1 and H9, NIH-designated
WA01 and WA09; passage 32– 40) (50) were expanded as previously
described (51) on an irradiated mouse embryonic fibroblast feeder layer
in growth medium consisting of knockout Dulbecco’s modified Eagle’s
medium, knockout serum replacement (KO-SR), L-glutamine, ␤-mercaptoethanol, nonessential amino acids, and human basic fibroblast
growth factor (Invitrogen, Carlsbad, CA). Cultures were split once a
week by incubation in 1 mg/ml collagenase IV (Invitrogen) for 10 min at
37 C and seeded on freshly prepared inactivated mouse embryonic fibroblasts. For formation of embryoid bodies, human ES colonies were
digested by using 1 mg/ml collagenase type IV and transferred into
ultra-low-attachment six-well plates (Corning Inc., Corning, NY) to
732
Xie et al.
Nuclear Receptors in ESCs
allow their aggregation in suspension. Human embryoid bodies were
grown in the same culture medium without human basic fibroblast
growth factor and with 20% fetal bovine serum (FBS) (Invitrogen) to
replace the KO-SR. Medium was changed every day. To test the undifferentiated state of human ESCs, colonies were fixed to analyze AKP
activity and ESC-specific surface markers, SSEA-4 and TRA-1-60,
through immunofluorescence using an ESC characterization kit following the manufacturer’s recommendations (Chemicon, Temecula, CA).
ESC differentiation in mouse and human ESCs was characterized by
QPCR using primers (see supplemental Table 4) to OCT4 and Nanog
(pluripotency primers) and three germ layer-specific proteins (AFP, nestin, GATA2, and keratin).
The murine ESC line (CMTI-1) was derived from the 129/SvEv/Tac
strain of mice and is widely used in generating germline transmission in
mice (Specialty Media, Flanders, NJ). These cells were routinely cultured on tissue culture plates coated with 0.1% gelatin (Sigma-Aldrich,
St. Louis, MO) in DMEM/F12 (Invitrogen) in the presence of 15% ES
cell-qualified FBS (Hyclone, Logan, UT), 0.1 mM 2-mercaptoethanol, 1
mM glutamine, 0.1 mM nonessential amino acids (Invitrogen) and 1000
U/ml murine leukemia inhibitory factor (Chemicon). Cells were
trypsinized and replated or re-fed every second day. Murine embryoid
bodies were formed from single mouse ESCs grown in suspension in
DMEM plus 10% ES cell-qualified FBS without leukemia inhibitory
factor on ultra-low-attachment six-well plates. Samples in the undifferentiated stage consisted of a minimum of 50 ESC clumps for human and
a minimum of 106 ESCs in suspension for mouse. Differentiated samples
contained 50 –100 embryoid bodies that were collected every day in
both cases.
RNA isolation and QPCR
Total cellular RNA was isolated from undifferentiated ESCs and
different time embryoid bodies using RNA Stat-60 (Tel-Test, Friendswood, TX) as previously described (21). The mRNA levels in each
sample were measured using the TaqMan-based standard curve assay
with an ABI 7900HT Sequence Detection System as described previously (52). The primer/probe sets for the 48 (human) and 49 (mouse)
NRs were validated as described (supplemental Table 2) (21) and online
at www.NURSA.org. PCR efficiencies were calculated according to the
previous report (21). PCR efficiencies were evaluated from the slope of
the standard curves using the formula E ⫽ 10⫺1/slope, where E is efficiency. The generated efficiency was used to convert Ct from log to
linear scale using E⫺Ct. Normalized mRNA levels were obtained by
dividing the averaged, efficiency-corrected nuclear receptor values by
that of 18S for each sample [(ENHR)⫺CtNHR/(E18S)⫺Ct18S]. The resulting
values were multiplied by 106 and plotted ⫾ SD from triplicates of each
sample. Ct for each NR was used to assess relative changes in mRNA
levels between two samples (52).
Bioinformatics analysis
Unsupervised cluster analysis was performed on the normalized
RNA levels by calculating Pearson’s centered correlation coefficients
followed by average linkage analysis using Eisen software (http://rana.
lbl.gov/eisen/). In brief, 1) pair-wise correlation values were calculated
for each receptor-to-receptor pair based on the tissue distribution pro¥共ai ⫺ a兲共bi ⫺ b兲
, where ai and bi are data
file, given the formula
冑共ai ⫺ a兲2 冑共bi ⫺ b兲2
points being compared and a and b are their respective averages. Then,
the correlation values were input into the Eisen software, which then
analyzed the data as follows. This calculation centers the data, meaning
that the scale of the y-axis is, in essence, ignored. 2) The resulting
Pearson coefficients were used to calculate the distance metric that is
illustrated by the lines connecting each member of a cluster on the heat
map. The NR pairs with the highest correlation coefficient segregated
together to form a node. The lengths of the lines between the nodes are
relative to the strength of their correlations.
冉
冊
Mol Endocrinol, May 2009, 23(5):724 –733
Acknowledgments
We thank Steven Kliewer for critically evaluating and reading the
manuscript.
Address all correspondence and requests for reprints to: David J.
Mangelsdorf, Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001
Forest Park Road, Dallas, Texas 75390-9041. E-mail: davo.mango@
utsouthwestern.edu; or Y. Eugene Chen, Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center,
Ann Arbor, Michigan 48109. E-mail: [email protected].
This work was supported by the Howard Hughes Medical Institute
(D.J.M.), Robert A. Welch Foundation (Grant I-1275 to D.J.M.), the
National Institutes of Health (NURSA Grant U19DK62434 to D.J.M.;
HL068878, HL075397, and HL89544 to Y.E.C.), the American Diabetes Association (7-03-JF-18 to M.F.), and the American Heart Association Southeast Affiliate (0525510B to C.-Q.X.). D.J.M. is an investigator of the Howard Hughes Medical Institute. Y.E.C. is an
established investigator of American Heart Association (0840025N).
Disclosure Summary: The authors have nothing to disclose.
References
1. Hoffman LM, Carpenter MK 2005 Characterization and culture of human
embryonic stem cells. Nat Biotechnol 23:699 –708
2. Donovan PJ, Gearhart J 2001 The end of the beginning for pluripotent stem
cells. Nature 414:92–97
3. Carter AM 2007 Animal models of human placentation: a review. Placenta
28(Suppl A):S41–S47
4. Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating the mouse
embryo: a laboratory manual. New York: Cold Spring Harbor Laboratory
Press
5. O’Rahilly R, Bossy J, Muller F 1981 [Introduction to the study of embryonic
stages in man]. Bull Assoc Anat (Nancy) 65:141–236 (French)
6. Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang
R, Guegler K, Rao MS, Mandalam R, Lebkowski J, Stanton LW 2004 Transcriptome characterization elucidates signaling networks that control human
ES cell growth and differentiation. Nat Biotechnol 22:707–716
7. Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, Brown MK,
Taylor DF, Steptoe AL, Wani S, Bethel G, Robertson AJ, Perkins AC, Bruce
SJ, Lee CC, Ranade SS, Peckham HE, Manning JM, McKernan KJ, Grimmond
SM 2008 Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5:613– 619
8. Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M,
Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS 2004 Differences between
human and mouse embryonic stem cells. Dev Biol 269:360 –380
9. Richards M, Tan SP, Tan JH, Chan WK, Bongso A 2004 The transcriptome
profile of human embryonic stem cells as defined by SAGE. Stem Cells 22:
51– 64
10. Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH 2003 Molecular signature of human embryonic stem cells and its comparison with the
mouse. Dev Biol 260:404 – 413
11. Wei CL, Miura T, Robson P, Lim SK, Xu XQ, Lee MY, Gupta S, Stanton L,
Luo Y, Schmitt J, Thies S, Wang W, Khrebtukova I, Zhou D, Liu ET, Ruan YJ,
Rao M, Lim B 2005 Transcriptome profiling of human and murine ESCs
identifies divergent paths required to maintain the stem cell state. Stem Cells
23:166 –185
12. Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ 2006
Anatomical profiling of nuclear receptor expression reveals a hierarchical
transcriptional network. Cell 126:789 –799
13. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ 2001 Nuclear receptors and
lipid physiology: opening the X-files. Science 294:1866 –1870
14. Germain P, Staels B, Dacquet C, Spedding M, Laudet V 2006 Overview of
nomenclature of nuclear receptors. Pharmacol Rev 58:685–704
15. Hong SH, Nah HY, Lee YJ, Lee JW, Park JH, Kim SJ, Lee JB, Yoon HS, Kim
CH 2004 Expression of estrogen receptor-␣ and -␤, glucocorticoid receptor,
and progesterone receptor genes in human embryonic stem cells and embryoid
bodies. Mol Cell 18:320 –325
16. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R 1985 The in vitro
development of blastocyst-derived embryonic stem cell lines: formation of
Mol Endocrinol, May 2009, 23(5):724 –733
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol
87:27– 45
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A 2000 Embryonic
stem cell lines from human blastocysts: somatic differentiation in vitro. Nat
Biotechnol 18:399 – 404
Murry CE, Keller G 2008 Differentiation of embryonic stem cells to clinically
relevant populations: lessons from embryonic development. Cell 132:661–
680
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A
2003 Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643– 655
Kuroda T, Tada M, Kubota H, Kimura H, Hatano SY, Suemori H, Nakatsuji
N, Tada T 2005 Octamer and Sox elements are required for transcriptional cis
regulation of Nanog gene expression. Mol Cell Biol 25:2475–2485
Fu M, Sun T, Bookout AL, Downes M, Yu RT, Evans RM, Mangelsdorf DJ
2005 A nuclear receptor atlas: 3T3-L1 adipogenesis. Mol Endocrinol 19:
2437–2450
Conley BJ, Denham M, Gulluyan L, Olsson F, Cole TJ, Mollard R 2005
Mouse embryonic stem cell derivation, and mouse and human embryonic
stem cell culture and differentiation as embryoid bodies. Curr Protoc Cell Biol
23:23.2.1–23.2.22
Rao M 2004 Conserved and divergent paths that regulate self-renewal in
mouse and human embryonic stem cells. Dev Biol 275:269 –286
Gupta P, Ho PC, Huq MM, Ha SG, Park SW, Khan AA, Tsai NP, Wei LN
2008 Retinoic acid-stimulated sequential phosphorylation, PML recruitment,
and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression. Proc
Natl Acad Sci USA 105:11424 –11429
Mullen EM, Gu P, Cooney AJ 2007 Nuclear receptors in regulation of mouse
ES cell pluripotency and differentiation. PPAR Res 2007:61563
Zhuang Y, Gudas LJ 2008 Overexpression of COUP-TF1 in murine embryonic stem cells reduces retinoic acid-associated growth arrest and increases
extraembryonic endoderm gene expression. Differentiation 76:760 –771
Fuhrmann G, Chung AC, Jackson KJ, Hummelke G, Baniahmad A, Sutter J,
Sylvester I, Schöler HR, Cooney AJ 2001 Mouse germline restriction of Oct4
expression by germ cell nuclear factor. Dev Cell 1:377–387
Honda M, Hamazaki TS, Komazaki S, Kagechika H, Shudo K, Asashima M
2005 RXR agonist enhances the differentiation of cardiomyocytes derived
from embryonic stem cells in serum-free conditions. Biochem Biophys Res
Commun 333:1334 –1340
Kastner P, Grondona JM, Mark M, Gansmuller A, LeMeur M, Decimo D,
Vonesch JL, Dollé P, Chambon P 1994 Genetic analysis of RXR␣ developmental function: convergence of RXR and RAR signaling pathways in heart
and eye morphogenesis. Cell 78:987–1003
Luo J, Sucov HM, Bader JA, Evans RM, Giguère V 1996 Compound mutants
for retinoic acid receptor (RAR)␤ and RAR␣1 reveal developmental functions
for multiple RAR␤ isoforms. Mech Dev 55:33– 44
Zechel C 2005 Requirement of retinoic acid receptor isotypes ␣, ␤, and ␥
during the initial steps of neural differentiation of PCC7 cells. Mol Endocrinol
19:1629 –1645
Duester G 2008 Retinoic acid synthesis and signaling during early organogenesis. Cell 134:921–931
Gajović S, Chowdhury K, Gruss P 1998 Genes expressed after retinoic acidmediated differentiation of embryoid bodies are likely to be expressed during
embryo development. Exp Cell Res 242:138 –143
Lammer EJ, Chen DT, Hoar RM, Agnish ND, Benke PJ, Braun JT, Curry CJ,
Fernhoff PM, Grix Jr AW, Lott IT, Richard JM, Sun SC 1985 Retinoic acid
embryopathy. N Engl J Med 313:837– 841
mend.endojournals.org
733
35. Thaller C, Eichele G 1987 Identification and spatial distribution of retinoids
in the developing chick limb bud. Nature 327:625– 628
36. Wichterle H, Lieberam I, Porter JA, Jessell TM 2002 Directed differentiation
of embryonic stem cells into motor neurons. Cell 110:385–397
37. Gu P, Goodwin B, Chung AC, Xu X, Wheeler DA, Price RR, Galardi C, Peng
L, Latour AM, Koller BH, Gossen J, Kliewer SA, Cooney AJ 2005 Orphan
nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development. Mol Cell Biol 25:3492–3505
38. Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun
Y, Lemischka IR 2006 Dissecting self-renewal in stem cells with RNA interference. Nature 442:533–538
39. Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH
2006 A protein interaction network for pluripotency of embryonic stem cells.
Nature 444:364 –368
40. van den Berg DL, Zhang W, Yates A, Engelen E, Takacs K, Bezstarosti K,
Demmers J, Chambers I, Poot RA 2008 Estrogen-related receptor ␤ interacts
with Oct4 to positively regulate Nanog gene expression. Mol Cell Biol 28:
5986 –5995
41. Zhang X, Zhang J, Wang T, Esteban MA, Pei D 2008 Esrrb activates Oct4
transcription and sustains self renewal and pluripotency in embryonic stem
cells. J Biol Chem 283:35825–35833
42. Chen J, Nathans J 2007 Estrogen-related receptor beta/NR3B2 controls epithelial cell fate and endolymph production by the stria vascularis. Dev Cell
13:325–337
43. Luo J, Sladek R, Bader JA, Matthyssen A, Rossant J, Giguère V 1997 Placental
abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-␤.
Nature 388:778 –782
44. Tremblay GB, Kunath T, Bergeron D, Lapointe L, Champigny C, Bader JA,
Rossant J, Giguère V 2001 Diethylstilbestrol regulates trophoblast stem cell
differentiation as a ligand of orphan nuclear receptor ERR␤. Genes Dev
15:833– 838
45. Burris TP, Guo W, McCabe ER 1996 The gene responsible for adrenal hypoplasia congenita, DAX-1, encodes a nuclear hormone receptor that defines a
new class within the superfamily. Recent Prog Horm Res 51:241–259; discussion 259 –260
46. Sun C, Nakatake Y, Ura H, Akagi T, Niwa H, Koide H, Yokota T 2008 Stem
cell-specific expression of Dax1 is conferred by STAT3 and Oct3/4 in embryonic stem cells. Biochem Biophys Res Commun 372:91–96
47. Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W
2000 Requirement for ␤-catenin in anterior-posterior axis formation in mice.
J Cell Biol 148:567–578
48. Park JI, Tsai SY, Tsai MJ 2003 Molecular mechanism of chicken ovalbumin
upstream promoter-transcription factor (COUP-TF) actions. Keio J Med 52:
174 –181
49. Warnecke M, Oster H, Revelli JP, Alvarez-Bolado G, Eichele G 2005 Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs
the functionality of the forebrain clock and affects nociception. Genes Dev
19:614 – 625
50. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M
2001 Insulin production by human embryonic stem cells. Diabetes 50:1691–
1697
51. Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA,
Itskovitz-Eldor J, Thomson JA 2000 Clonally derived human embryonic stem
cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278
52. Bookout AL, Mangelsdorf DJ 2003 Quantitative real-time PCR protocol for
analysis of nuclear receptor signaling pathways. Nucl Recept Signal 1:e012