L1TD1 Is a Marker for Undifferentiated Human
Embryonic Stem Cells
Raymond Ching-Bong Wong1,3, Abel Ibrahim1,3, Helen Fong1,3, Noelle Thompson1,3, Leslie F. Lock1,2,3,
Peter J. Donovan1,2,3*
1Department of Biological Chemistry, University of California Irvine, Irvine, California, United States of America, 2Department of Developmental and Cell Biology,
University of California Irvine, Irvine, California, United States of America, 3Sue and Bill Gross Stem Cell Research Centre, University of California Irvine, Irvine, California,
United States of America
Background: Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the
three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating
stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In
particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many
of the downstream targets of these transcription factors are not well characterized. Furthermore, it remains unknown
whether additional novel stem cell factors are involved in the establishment and maintenance of the stem cell state.
Methodology/Principal Findings: Here we show that a novel gene, L1TD1 (also known as FLJ10884 or ECAT11), is
abundantly expressed in undifferentiated hESC. Differentiation of hESC via embryoid body (EB) formation or BMP4
treatment results in the rapid down-regulation of L1TD1 expression. Furthermore, populations of undifferentiated and
differentiated hESC were sorted using the stem cell markers SSEA4 and TRA160. Our results show that L1TD1 is enriched in
the SSEA4-positive or TRA160-positive population of hESC. Using chromatin immunoprecipitation we found enriched
association of Nanog to the predicted promoter region of L1TD1. Furthermore, siRNA-mediated knockdown of Nanog in
hESC also resulted in downregulation of L1TD1 expression. Finally, using luciferase reporter assay we demonstrated that
Nanog can activate the L1TD1 upstream promoter region. Altogether, these results provide evidence that L1TD1 is a
downstream target of Nanog.
Conclusion/Significance: Taken together, our results suggest that L1TD1 is a downstream target of Nanog and represents a
useful marker for identifying undifferentiated hESC.
Citation: Wong RC-B, Ibrahim A, Fong H, Thompson N, Lock LF, et al. (2011) L1TD1 Is a Marker for Undifferentiated Human Embryonic Stem Cells. PLoS ONE 6(4):
Editor: Henning Ulrich, University of Sa ˜o Paulo, Brazil
Received December 16, 2010; Accepted March 31, 2011; Published April 29, 2011
Copyright: ? 2011 Wong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the California Institute of Regenerative Medicine (http://www.cirm.ca.gov/; T1-00008 for RC-BW and HF, TG2-01152 for NT,
RCI-00110 to PJD) and the University of California Irvine startup funds to LFL. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Human embryonic stem cells (hESC) are pluripotent cells that
can self-renew indefinitely and also generate cells representative of
the three primary embryonic germ layers [1,2]. The latter ability,
termed pluripotency, makes hESC an ideal tool to develop cell
replacement therapies. However, before the therapeutic potential
of hESC can be fully realized, development of a culture system
that enhances the production of undifferentiated hESC will be
essential. Studies of the molecular mechanisms regulating hESC
pluripotency could be helpful in this regard. Several transcription
factors are known to be important regulators of pluripotency and
self-renewal in hESC, including Oct4, Sox2 and Nanog [3,4,5,6].
These three transcription factors are able to regulate the
expression of each other, effectively forming a core transcriptional
network governing pluripotency of hESC . A similar transcrip-
tional network between Tcl1, Tbx3 and Esrrb also exist in mouse
embryonic stem cells (mESC) . However, whether there are
other factors involved in regulating hESC pluripotency remains to
be determined. Studies that address these questions could provide
critical insights into the mechanisms regulating self-renewal and
early differentiation of hESC.
We are interested in the novel gene FLJ10884, which was
recently renamed as LINE 1 type transposase domain containing 1
(L1TD1) in the NCBI database (http://www.ncbi.nlm.nih.gov).
Moreover, it was categorized as ECAT11, a member of the
Embryonic Stem Cell Associated Transcripts (ECAT). The ECAT
genes are a set of genes that were found to be enriched in mESC
compared to somatic cells using a digital differential display
method . Notably, many ECAT genes have been found to play
an important role in stem cell biology. For instance, ECAT4 was
identified to be Nanog, a master regulator for the maintenance of
mESC and hESC [3,9]. ECAT5 was identified to be Embryonic
stem cell expressed Ras (ERas), a Ras-like oncogene that is
important in regulating mESC proliferation . ECAT9 was
identified as Growth and differentiation factor 3 (GDF3), an
PLoS ONE | www.plosone.org1April 2011 | Volume 6 | Issue 4 | e19355
important factor that helps maintain mESC pluripotency by
inhibiting bone morphogenetic protein (BMP) signaling .
In this study, we have characterized the expression of L1TD1 in
hESC. We showed that L1TD1 is highly enriched in undifferen-
tiated hESC compared to their differentiated derivatives, indicat-
ing that it may be a useful marker to identify undifferentiated
hESC. Moreover, we demonstrated that L1TD1 is a target gene of
L1TD1 is a marker for undifferentiated hESC
Using quantitative PCR, we compared the mRNA expression
levels of L1TD1 in undifferentiated and differentiated hESC.
hESC were differentiated by two methods: differentiation by
embryoid body (EB) formation, or differentiation by BMP4
treatment . Our results demonstrate that L1TD1 mRNA is
rapidly downregulated upon EB formation (Figure 1A). In day 5
EB, L1TD1 expression is downregulated to 56617% of the level
in undifferentiated hESC. By day 21, L1TD1 mRNA is almost
undetectable (0.4% of the level in undifferentiated hESC). This
expression pattern of L1TD1 observed upon EB differentiation is
similar to other known pluripotent markers including Oct4, Sox2
and Nanog (Figure 1A). Similar results were observed with mESC,
in which EB differentiation also led to downregulation of L1TD1
transcripts (Figure 1E). Notably, mouse embryonic fibroblast
(MEF) feeders do not express the L1TD1 gene (Figure 1E). Next,
we differentiated hESC using BMP4 treatment. This also resulted
in downregulation of L1TD1 mRNA, in a trend similar to the
downregulation of Oct4, Sox2 and Nanog (Figure 1B). hESC
treated with BMP4 for 5 days displayed 4167% of the level of
L1TD1 expression in comparison to untreated hESC. By day 7
following BMP4 treatment, the level of L1TD1 dropped down to
only 1363% compared to untreated hESC (Figure 1B). Therefore
our data suggests that L1TD1 mRNA is rapidly downregulated
upon hESC differentiation. Similar results are observed in another
cell line H1 (Figure S1A).
Since residual undifferentiated hESC may remain even after
prolonged differentiation following EB formation or BMP4
treatment, this may introduce undesired variability in our
quantitative PCR results. To address this issue of heterogeneity,
we used fluorescence-activated cell sorting (FACS) to separate
undifferentiated and differentiated hESC populations using the
known stem cell markers TRA160 and SSEA4. In addition, we
excluded the MEF feeders from our sample using antibodies to the
MEF marker Thy1.2 . As illustrated in Figure 1C, our results
showed that L1TD1 mRNA is downregulated in SSEA4-negative
hESC (563% of SSEA4 positive-hESC) or TRA160-negative
hESC (362% of TRA160-positive hESC). An analysis of Oct4,
Sox2 or Nanog mRNA levels in SSEA4-negative or TRA160-
negative hESC showed similar downregulation of these factors as
expected. Similar results were obtained for hESC sorted with
TRA181, an antibody that recognizes different epitopes of the
same antigen as TRA160 (data not shown). We have also
confirmed the results in two different hESC line, H9 (Figure 1C)
and H1 (Figure S1B), supporting the notion that L1TD1 can be
used to identify undifferentiated hESC in multiple cell lines.
Since a small portion of the SSEA4-negative or TRA160-
negative hESC retain expression of L1TD1 (Figure 1C), we sought
to determine what lineage of differentiated hESC tends to retain
L1TD1. In Figure 1D, we showed that hESC promoted to
differentiate along the neural lineage do not express L1TD1, as we
failed to detect L1TD1 expression in hESC-derived neural
progenitor, and cells promoted to differentiate into oligodendro-
cytes, astrocytes and neurons. In silico analysis of L1TD1
expression using the EST profile viewer from the Unigene
(http://www.ncbi.nlm.nih.gov/unigene) revealed that L1TD1 is
absent in many adult tissues, except for low expression level in
blood tissues, connective tissues, placenta and the testis (Figure S2).
Thus, we cannot rule out the possibility that hESC differentiated
into these tissues may retain low expression of L1TD1.
To study the protein expression of L1TD1 in hESC, we
generated an anti-L1TD1 antibody in rabbits against a synthetic
peptide corresponding to the C-terminal sequence of human
L1TD1 (amino acids 686–699). Using western blot analysis,
L1TD1 protein is detected as a ,100 kDa band in undifferen-
tiated hESC (Figure 2), similar to the expected molecular size of
L1TD1 calculated base on its amino acid sequence (,98 kDa).
However, such band is not detectable in Day 14 EB (Figure 2).
Two other bands (,37 kDa and ,40 kDa) are also detected in
some experiments but it is not consistently detected. Furthermore,
pre-incubation of the anti-L1TD1 antibody with the peptide
antigen eliminated all bands, suggesting that the anti-L1TD1
antibody is specific to the designed sequence of human L1TD1
gene (Figure 2). However, this anti-L1TD1 antibody did not work
for immunocytochemistry (data not shown). Nevertheless, our
results suggest that L1TD1 protein is abundant in undifferentiated
hESC and downregulated upon differentiation, rendering it a
useful marker for undifferentiated hESC.
L1TD1 is a downstream target for Nanog
Using the Genomatix program (http://www.genomatix.de), we
identified a Nanog binding site (AATG) ,280 bp upstream of the
transcription start site in the predicted promoter region of L1TD1
(Figure 3A). Therefore, we analyzed the association of Nanog to
the L1TD1 gene in undifferentiated hESC by chromatin
immunoprecipitation (ChIP). Using quantitative PCR, our results
demonstrated a 10.8-fold enrichment of Nanog association to the
upstream region of L1TD1 in undifferentiated hESC compared to
the isotype-matched control, suggesting L1TD1 is a downstream
target of Nanog (Figure 3B). No band is observed in our negative
controls using samples immunoprecipitated in the absence of
antibody or with an isotype antibody, indicating the immunopre-
cipitation procedure of our ChIP assay is specific. Moreover, we
carried out siRNA-mediated knockdown of Nanog in hESC. As
shown in Figure 3C, knockdown of Nanog expression levels
(37612% of mock transfected hESC) resulted in a differentiated
morphology in hESC, as well as downregulation of L1TD1
transcripts (42616% of mock transfected hESC). We also showed
that knockdown of a control gene ß2M has no effect on the
expression of L1TD1 (Figure 3C).
To further study the interaction between Nanog and the
L1TD1 gene, we performed luciferase assay by constructing a
luciferase reporter vector driven by the upstream promoter region
of L1TD1 (pL1TD1-luc). As hESC possess high level of
endogenous Nanog expression, we opted to perform the luciferase
assay in SW480 cells, a human colon adenocarcinoma cell line
without detectable level of endogenous Nanog expression (data not
shown, and ), in order to minimize background noise of
luciferase reading. Our luciferase assay results demonstrated that
ectopic expression of Nanog activates the L1TD1 upstream
promoter region, resulting in a 6.8 fold increase in luciferase
activity compared to control (Figure 3D). We verified that this
increase in luciferase activity is specific to the interaction between
Nanog and the L1TD1 upstream promoter region, as we observed
low luciferase activity in our negative controls without the L1TD1
upstream promoter region or with ectopic expression of GFP
(Figure 3D). Altogether, our results from luciferase assays are
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consistent with our ChIP and Nanog knockdown studies,
providing evidence that L1TD1 is a downstream target of Nanog.
Here we have characterized the expression of a novel gene
L1TD1 in hESC. Using quantitative PCR and western blot
analysis, we demonstrated that L1TD1 are highly expressed in
hESC and rapidly downregulated upon differentiation of hESC by
EB formation (Figure 1A and 2) or BMP4 treatment (Figure 1B).
Furthermore, L1TD1 expression is downregulated in differentiat-
ed SSEA4-negative or TRA160-negative hESC (Figure 1C and
Figure S1B). This result is consistent with a previous DNA
microarray study that demonstrated L1TD1 is also enriched in
undifferentiated hESC . Furthermore, we provided evidence
that the L1TD1 protein expression is also downregulated upon
hESC differentiation in multiple hESC cell lines. Therefore,
L1TD1 can be used as a marker to distinguish undifferentiated
hESC from differentiated progenitors and the MEF feeders
(Figure 1 and 2).
Our in silico analysis indicated that L1TD1 expression is highly
enriched during the blastocyst stage of embryogenesis and absent
in many adult tissues in human (Figure S2), an expression pattern
much like that observed for Nanog . Therefore, we wondered
whether Nanog could regulate L1TD1 in hESC. In this study, we
showed that Nanog is associated with a site situated in the
upstream predicted promoter region of L1TD1, suggesting that
L1TD1 expression may be regulated by Nanog (Figure 3B). Using
luciferase reporter assay, we provided evidence suggesting that
Nanog can bind to the upstream promoter region of L1TD1 and
activate its expression (Figure 3D). Consistent with this idea, we
also demonstrated that Nanog knockdown in hESC resulted in
downregulation of L1TD1 expression (Figure 3C).
At the moment, no previous study has addressed the functional
role of L1TD1. Our in silico analysis revealed a full Transposase 22
domain near the C terminus and another truncated Transposase
22 domain in human L1TD1 (data not shown). A previous study
has described the Transposase 22 domain as a misnomer and that
this domain does not encode for a transposase, but actually
represents the open reading frame (ORF) 1 protein of the Long
interspersed element-1 (L1) retrotransposon , also known as
leucine zipper protein p40 . A functional L1 element encodes
for two proteins, ORF1 protein and ORF2 protein (reviewed in
). Whereas ORF2 encodes for a protein with endonuclease
and reverse transcriptase activity [19,20,21], less is known about
the role of ORF1. Mutagenesis studies indicated that ORF1 is
required for L1 retrotransposition [21,22]. In this aspect, a
previous study found that undifferentiated hESC can support L1
retrotransposition in vitro , thus L1TD1 may play a role in
regulating L1 retrotransposition in hESC. Also, the ORF1 protein
possess RNA-binding [24,25] and RNA chaperone activity .
Therefore, it is possible that L1TD1 may exhibit similar functions
as well. Moreover, a yeast-two-hybrid screen revealed that ORF1
protein can bind to components of the RNA-induced silencing
(RISC) complex involved in RNA interference, as well as other
proteins involved in mRNA transport . Given that L1TD1
protein contain the Transposase 22 domain/L1 ORF1, it is
possible that L1TD1 can bind to the same binding partners as L1
Figure 2. Protein expression of L1TD1 in undifferentiated and differentiated hESC. Left) Western blot analysis of L1TD1 in undifferentiated
H1, H9 and H14 cells and the corresponding Day 14 EB. 2 mg of peptide antigen for generation of the L1TD1 antibody was used as a positive control.
Right) Peptide blocking of the L1TD1 antibody was performed to ensure the specificity of the L1TD1 antibody.
Figure 1. mRNA expression of L1TD1 in undifferentiated and differentiated hESC. Quantitative PCR of L1TD1, Oct4, Sox2 and Nanog
expression in undifferentiated hESC compared to A) EB or B) hESC treated with BMP4 at different time points. Average relative expression levels and
standard deviations from three quantitative PCR reactions for each sample are shown. Representative results from H9 are shown. C) Flow cytogram of
hESC sorted with TRA160 or SSEA4. Negative isotype controls were used to set the gating for Thy1.2, TRA160 or SSEA4. Quantitative PCR of L1TD1,
Oct4, Sox2 and Nanog expression was carried out comparing SSEA4+ hESC with SSEA42 hESC (Top panel), or TRA160+ hESC with TRA1602 hESC
(bottom panel). Average relative expression levels and standard deviations from three quantitative PCR reactions for each sample are shown.
Representative results from H9 are shown. D) Quantitative PCR of L1TD1 expression in undifferentiated hESC, neural progenitors, and cells promoted
to differentiate into oligodendrocytes, astrocytes and neurons (n=2). E) Quantitative PCR of L1TD1 expression in MEF, undifferentiated mESC and EB
at different time points. Average relative expression levels and standard deviations from three quantitative PCR reactions for each sample are shown.
Representative results from R1 are shown and biological repeats were carried out in GSI1 mESC lines (Data not shown).
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Figure 3. L1TD1 is a downstream target of Nanog. A) Schematic diagram of the L1TD1 gene structure showing the presence of a Nanog
binding site in the predicted promoter region. Exons are depicted as boxes and the coding region of L1TD1 are shaded. B) ChIP results of H9 sample
immunoprecipitated with antibody to Nanog. Negative controls were performed with samples immunoprecipitated in the absence of antibody or
with an isotype-matched antibody. The input is a positive control of samples prior to immunoprecipitation. The picture is a representative result from
L1TD1 Is a hESC Marker
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ORF1. Clearly, further research is needed to evaluate the precise
function of L1TD1 in hESC. In summary, we have demonstrated
that the novel gene L1TD1 can be used as a marker for
undifferentiated hESC and is a downstream target of Nanog.
Materials and Methods
hESC cell lines H1, H9 and H14 (WiCell) were cultured on
mitotically-inactivated MEF (Millipore) and media supplemented
with 20% knockout serum replacement and 4 ng/ml bFGF
(Invitrogen) as described by Xu et al. (2001) . Medium was
changed every day and cells were passaged using 1 mg/ml
collagenase IV (Invitrogen) every 7 days. hESC were maintained
in an incubator at 37uC with 5% CO2. In some experiments,
hESC were treated with 25 ng/ml of BMP4 (R&D Systems) to
induce differentiation as described by Pera et al. . EB were
formed by growing hESC in suspension in a low attachment
culture plate with media supplemented with 20% knockout serum
replacement. H9 hESC-derived neural progenitor cells (Millipore)
were maintained in ENStem-A neural expansion medium
(Millipore) on plates pre-coated with poly-l-ornithine (Sigma)
and laminin (Sigma), according to manufacturer’s instructions.
hESC-derived neural progenitor cells were differentiated into
oligodendrocytes, neurons and astrocytes with protocols described
in . Briefly, hESC-derived neural progenitor cells were
cultured in DMEM supplemented with N1 (Invitrogen) and
PDGF-A (2 ng/ml, Peprotech) for 8 days to promote oligoden-
drocyte differentiation. Alternatively, the neural progenitor cells
were promoted to differentiate into neurons and astrocytes by
culturing on ornithine/laminin substrate for 8 days with medium
consisting of DMEM/F12, N2 supplement (Invitrogen), cAMP
(100 ng/ml, Sigma), and brain-derived neurotrophic factor
(BDNF, 10 ng/ml; PeproTech). mESC cell lines R1 and GSI1
were cultured in media containing leukemia inhibitory factor (LIF,
Millipore, 1000Units/ml) and 16% fetal calf serum (Hyclone). R1
mESC were cultured in feeder-free conditions whereas GSI1
mESC were cultured in the presence of MEF. EB were formed by
growing mESC in suspension in low attachment culture plates in
media without LIF. SW480 cells were maintained in DMEM
supplemented with 10% fetal calf serum (Hyclone), 1% L-
glutamine and 0.5% penicillin/streptomycin (All from Invitrogen).
RNA samples were extracted from H1, H9 and H14 cells using
an RNeasy kit (Qiagen). cDNA was synthesized from 1 mg RNA
usingthe high capacity cDNA
(Applied Biosystems). Taqman mastermix and probes (Oct4:
Hs00999632_g1; Sox2: Hs01053049_s1; Nanog: Hs02387400_g1;
L1TD1: Hs00219458_m1; 18S: Hs99999901_s1) were all pur-
chased from Applied Biosystems. Quantitative PCR was performed
following the manufacturer’s instructions.Samples were analyzed in
a 7900HT qPCR machine (Applied biosystems). Briefly, samples
were run in a 384 well plates with the following thermal profile:
95uC for 10 minutes, followed by 40 cycles of 95uC for 15 seconds
and 60uC for 1 minute. The Ct threshold was set using the
parameters by the SDS software (Applied biosystems) and
subsequently confirmed manually to obtain the appropriate Ct
value. The results were normalised to the housekeeping gene 18S
and analysed using the DDCt method as described by Bookout et al.
Fluorescence-activated cell sorting (FACS)
H1 and H9 cells were harvested and dissociated with 0.25%
trypsin/EDTA (Invitrogen). The samples were blocked in 10%
serum and immunostained with TRA160, TRA181 (Santa Cruz)
or SSEA4 (Caltag laboratories) followed by an Alexa-fluor 488
secondary antibody (Invitrogen). Finally, the samples were stained
with the PE-conjugated Thy1.2 antibody (BD Pharmingen) to
allow sorting of the MEF from hESC . Samples were sorted
using a flow cytometer (MoFlo). Isotype-matched controls were
used to set the gating for FACS sorting.
A polyclonal antibody was generated in rabbits against a
synthetic peptide corresponding to amino acids 686–699 of human
L1TD1 (SKERQRDIEERSRS) (Genescript). An N-terminal
cysteine was added to the peptide to allow it to conjugate with
Keyhole Limpet Hemocyanin (KLH). Two separate rabbits were
immunized with the peptide antibody on day 1, 14, 35 and 56.
Anti-sera were collected 14 days after the final immunisation. The
antiserum was affinity-purified using the SulfoLink immobilization
kit following manufacturer’s instructions (Thermo Scientific).
Briefly, the peptide antigen is coupled with Sulfolink resin to
make an affinity column. Ten ml of antiserum was repeatedly run
through the column, and the purified antibodies were eluted in
buffer containing sodium azide.
Western blot analysis
H1, H9 and H14 cells were lysed in Radioimmunoprecipitation
(RIPA) buffer and sample reducing buffer containing ß-mercap-
toethanol as described previously . Briefly, samples were run in
a 4% stacking gel and 10% resolving gel and transferred to a
PVDF membrane (GE Healthcare). The membrane was blotted
with anti-L1TD1 antibody (1:2000) followed by incubation with a
goat anti-rabbit horseradish peroxide (HRP)-conjugated secondary
antibody. Peptide blocking is performed by pre-incubation of the
anti-L1TD1 antibody with the synthetic peptide antigen (60 mg)
for 2 to 4 hours at room temperature or overnight at 4uC prior to
immunoblotting. Chemilluminescent detection reagent (ECL plus,
GE Healthcare) was used to detect the HRP signal on film or using
the Gel-doc system (Biorad). Subsequently, the membrane was
stripped with the Restore western blot stripping buffer (Thermo
scientific) and re-blotted with an anti-ß-actin antibody (Santa
Cruz) and the appropriate HRP secondary antibody.
Chromatin immunoprecipitation (ChIP)
ChIP assays with H9 cells were carried out as described
previously by Zeng et al. . Briefly, 66106cells were crosslinked
with 1% formaldehyde at 37uC for 10 minutes. The formaldehyde
was neutralized by the addition of 125 mM glycine and the cells
three independent experiments. Quantitative PCR is performed to quantify the ChIP samples. Average relative expression levels and standard
deviations from three independent experiments are shown. C) Morphology of hESC nucleofected with siRNA against Nanog, ß2M and a mock control
(Top panel). Quantitative PCR of L1TD1 and Nanog expression in hESC nucleofected with siRNA against Nanog, ß2M and a mock control (Bottom
panel). Average relative expression levels and standard deviations from three independent experiments are shown. D) Luciferase assay of SW480 co-
transfected with different combination of the following vectors: a promoterless luciferase reporter vector (pEMPTY-luc), a luciferease reporter vector
driven by the L1TD1 upstream promoter region (pL1TD1-luc), an overexpression vector for GFP (pmaxGFP) or Nanog (pCMV-Nanog). Average relative
luciferase activity and standard deviations from three independent experiments are shown.
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were lysed with SDS lysis buffer. The nuclear extracts containing
DNA fragments of about 500 base pairs were pre-cleared with
protein A sepharose beads (GE Healthcare) previously incubated
with 1 mg/ml of bovine serum albumin (BSA, Sigma) and
0.2 mg/ml of salmon sperm DNA (Ambion) at 4uC for
20 minutes. Four to eight micrograms of anti-Nanog polyclonal
antibody (R&D Systems) were then incubated with the nuclear
extracts at 4uC overnight and precipitated with protein A
sepharose beads (GE Healthcare). The complex was washed and
eluted with 1% SDS and 100 mM NaHCO3. Crosslinks were
reversed by incubating at 65uC for four to six hours and the DNA
was recovered using a gel cleanup kit (Eppendorf). PCR was
performed with the Taq PCR core kit (Qiagen) using primers
targeting the Nanog binding site upstream of L1TD1 (Forward:
CGCCTCATCAC; annealing temperature 51uC). PCR products
were electrophoresed in a 1% agarose gel and imaged using the
Gel-Doc Imager (Bio-Rad). Alternatively, quantitative PCR is
performed using Sybr green mastermix (Applied biosystems) with
the same primer set. A standard curve using cDNA with 5 fold
dilutions was constructed to ensure high PCR efficiency and a
dissociation curve is constructed to ensure specific target
amplification. Quantitative PCR was performed in triplicate in
384 well plates using thermal profile specified by the manufactur-
er. Samples were analyzed in a 7900HT qPCR machine (Applied
biosystems). The results were analysed using the DDCt method as
described by Bookout et al. .
siRNA-mediated knockdown of Nanog
Nanog knockdown in H9 cells was carried out using nucleofec-
tion, following procedures described in [4,6]. Briefly, H9 cells were
trypsinized and nucleofected with 100 nM of siRNA targeting
Nanog (AAGGGTTAAGCTGTAACATAC). Cells nucleofected
in the absence of siRNA or with 100 nM of siRNA targeting Beta-
2-microglobulin (ß2M) were used as negative controls (AAGATT-
CAGGTTTACTCACGT). Following nucleofection, the cells
were plated on MEF and cultured in the presence of neurotro-
phins (50 ng/ml each of BDNP, NT3 and NT4). RNA samples
were harvested 4 days after nucleofection and knockdown of
Nanog expression was confirmed by quantitative PCR.
Luciferase reporter assay
Luciferase reporter plasmid driven by the L1TD1 upstream
promoter region (pL1TD1-luc) is constructed by inserting the
L1TD1 upstream promoter region (21230 To +3) into the
promoterless pGL4.10 plasmid (Promega). The L1TD1 upstream
region is cloned using the following primers: Forward primer: 59-
GGGGAGTTTGGCTCCTGTAGA-39; Reverse primer: 59-AA-
GGACTGAGAGGATTCCCGATC-39. A promoterless pGL4.10
plasmid (pEMPTY-luc) is used as a negative control to determine
background luciferase activity. The plasmid pCMV-Nanog
(Origene) is used to overexpress Nanog, alternatively pmaxGFP
(Lonza) is used to express GFP as stuffer DNA to gauge
transfection efficiency. SW480 were cultured in 12-well plates
and co-transfected with the corresponding luciferase reporter
vector (100 ng/well) in the presence of pCMV-Nanog or
pmaxGFP (1 mg/well) using Express-in (Open biosystems). Cells
were harvested 24 hours post-transfection and assay for luciferase
activity using the Luciferase reporter assay system (Promega)
following manufacturer’s instructions. Samples were measured
using a luminometer (Berthold Technologies). The luciferase
activities were then normalized to cell number by quantification of
the total protein concentration in the samples using the Non-
interfering protein assay (GBiosciences) following manufacturer’s
differentiation is observed in different cell line. A)
Quantitative PCR of L1TD1 expression in undifferentiated H1
compared to EB at different time points. Average relative
expression levels and standard deviations from three quantitative
PCR reactions for each sample are shown. B) Quantitative PCR of
L1TD1 expression in H1 sorted with TRA160 or SSEA4. Average
relative expression levels and standard deviations from three
quantitative PCR reactions for each sample are shown.
L1TD1 mRNA downregulation upon hESC
in different tissues. The expression profile of L1TD1 in
different tissue samples was extracted from the Unigene database.
The abundance of L1TD1 transcripts in different tissue samples
are presented as the number of EST transcripts per million (TPM),
a value that normalized the L1TD1 EST counts to the total EST
counts of the sample.
In silico study of expression profile of L1TD1
Conceived and designed the experiments: RC-BW PJD. Performed the
experiments: RC-BW AI HF NT. Analyzed the data: RC-BW AI HF.
Contributed reagents/materials/analysis tools: PJD LFL. Wrote the paper:
RC-BW. Review and final approval of manuscript: RC-BW AI HF PJD
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al.
(1998) Embryonic stem cell lines derived from human blastocysts. Science 282:
2. 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.
3. Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, et al. (2005)
Downregulation of NANOG induces differentiation of human embryonic stem
cells to extraembryonic lineages. Stem Cells 23: 1035–1043.
4. Fong H, Hohenstein KA, Donovan PJ (2008) Regulation of self-renewal and
pluripotency by Sox2 in human embryonic stem cells. Stem Cells 26: 1931–1938.
5. Matin MM, Walsh JR, Gokhale PJ, Draper JS, Bahrami AR, et al. (2004)
Specific knockdown of Oct4 and beta2-microglobulin expression by RNA
interference in human embryonic stem cells and embryonic carcinoma cells.
Stem Cells 22: 659–668.
6. Hohenstein KA, Pyle AD, Chern JY, Lock LF, Donovan PJ (2008)
Nucleofection mediates high-efficiency stable gene knockdown and transgene
expression in human embryonic stem cells. Stem Cells 26: 1436–1443.
7. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. (2005) Core
transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:
8. Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, et al. (2006) Dissecting self-
renewal in stem cells with RNA interference. Nature 442: 533–538.
9. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, et al. (2003) The
homeoprotein Nanog is required for maintenance of pluripotency in mouse
epiblast and ES cells. Cell 113: 631–642.
10. Takahashi K, Mitsui K, Yamanaka S (2003) Role of ERas in promoting tumour-
like properties in mouse embryonic stem cells. Nature 423: 541–545.
11. Levine AJ, Brivanlou AH (2006) GDF3, a BMP inhibitor, regulates cell fate in
stem cells and early embryos. Development 133: 209–216.
12. Pera MF, Andrade J, Houssami S, Reubinoff B, Trounson A, et al. (2004)
Regulation of human embryonic stem cell differentiation by BMP-2 and its
antagonist noggin. J Cell Sci 117: 1269–1280.
13. Laslett AL, Grimmond S, Gardiner B, Stamp L, Lin A, et al. (2007)
Transcriptional analysis of early lineage commitment in human embryonic
stem cells. BMC Dev Biol 7: 12.
L1TD1 Is a hESC Marker
PLoS ONE | www.plosone.org7April 2011 | Volume 6 | Issue 4 | e19355
14. Kohler EE, Cowan CE, Chatterjee I, Malik AB, Wary KK (2011) NANOG Download full-text
induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell
proliferation and angiogenesis. Blood 117: 1761–1769.
15. Enver T, Soneji S, Joshi C, Brown J, Iborra F, et al. (2005) Cellular
differentiation hierarchies in normal and culture-adapted human embryonic
stem cells. Hum Mol Genet 14: 3129–3140.
16. Martin SL (2006) The ORF1 Protein Encoded by LINE-1: Structure and
Function During L1 Retrotransposition. J Biomed Biotechnol 2006: 45621.
17. Holmes SE, Singer MF, Swergold GD (1992) Studies on p40, the leucine zipper
motif-containing protein encoded by the first open reading frame of an active
human LINE-1 transposable element. J Biol Chem 267: 19765–19768.
18. Goodier JL, Kazazian HH, Jr. (2008) Retrotransposons revisited: the restraint
and rehabilitation of parasites. Cell 135: 23–35.
19. Feng Q, Moran JV, Kazazian HH, Jr., Boeke JD (1996) Human L1
retrotransposon encodes a conserved endonuclease required for retrotranspo-
sition. Cell 87: 905–916.
20. Mathias SL, Scott AF, Kazazian HH, Jr., Boeke JD, Gabriel A (1991) Reverse
transcriptase encoded by a human transposable element. Science 254:
21. Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, et al. (1996) High
frequency retrotransposition in cultured mammalian cells. Cell 87: 917–927.
22. Martin SL, Cruceanu M, Branciforte D, Wai-Lun Li P, Kwok SC, et al. (2005)
LINE-1 retrotransposition requires the nucleic acid chaperone activity of the
ORF1 protein. J Mol Biol 348: 549–561.
23. Garcia-Perez JL, Marchetto MC, Muotri AR, Coufal NG, Gage FH, et al.
(2007) LINE-1 retrotransposition in human embryonic stem cells. Hum Mol
Genet 16: 1569–1577.
24. Hohjoh H, Singer MF (1997) Sequence-specific single-strand RNA binding
protein encoded by the human LINE-1 retrotransposon. EMBO J 16:
25. Kolosha VO, Martin SL (2003) High-affinity, non-sequence-specific RNA
binding by the open reading frame 1 (ORF1) protein from long interspersed
nuclear element 1 (LINE-1). J Biol Chem 278: 8112–8117.
26. Martin SL, Bushman FD (2001) Nucleic acid chaperone activity of the ORF1
protein from the mouse LINE-1 retrotransposon. Mol Cell Biol 21: 467–475.
27. Goodier JL, Zhang L, Vetter MR, Kazazian HH, Jr. (2007) LINE-1 ORF1
protein localizes in stress granules with other RNA-binding proteins, including
components of RNA interference RNA-induced silencing complex. Mol Cell
Biol 27: 6469–6483.
28. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, et al. (2001) Feeder-free
growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:
29. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro
differentiation of transplantable neural precursors from human embryonic stem
cells. Nat Biotechnol 19: 1129–1133.
30. Bookout AL, Mangelsdorf DJ (2003) Quantitative real-time PCR protocol for
analysis of nuclear receptor signaling pathways. Nucl Recept Signal 1: e012.
31. Wong RC, Tellis I, Jamshidi P, Pera M, Pebay A (2007) Anti-apoptotic effect of
sphingosine-1-phosphate and platelet-derived growth factor in human embry-
onic stem cells. Stem Cells Dev 16: 989–1001.
32. Zeng W, de Greef JC, Chen YY, Chien R, Kong X, et al. (2009) Specific loss of
histone H3 lysine 9 trimethylation and HP1gamma/cohesin binding at D4Z4
repeats is associated with facioscapulohumeral dystrophy (FSHD). PLoS Genet
L1TD1 Is a hESC Marker
PLoS ONE | www.plosone.org8 April 2011 | Volume 6 | Issue 4 | e19355