EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
LacdiNAc (GalNAcb1-4GlcNAc) Contributes to Self-Renewal of
Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory
Factor/STAT3 Signaling
NORIHIKO SASAKI,a MASAHITO SHINOMI,a KAZUMI HIRANO,a KUMIKO UI-TEI,b SHOKO NISHIHARAaaLaboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji,
Tokyo, Japan; bBiophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan
Key Words. LacdiNAc • Leukemia inhibitory factor/STAT3 signaling • Embryonic stem cell • Self-renewal

ABSTRACT
Self-renewal of mouse embryonic stem cells (mESCs) is
maintained by leukemia inhibitory factor (LIF)/signaltransducer and activator of transcription (STAT3) signaling. However, this signaling control does not function inneither mouse epiblast stem cells (mEpiSCs) nor human
ESCs (hESCs) or human induced pluripotent stem cells(hiPSCs). To date, the underlying molecular mechanismsthat determine this differential LIF-responsiveness have notbeen clariﬁed. Here, we show that the cell surface glycan
LacdiNAc (GalNAcb1-4GlcNAc) is required for LIF/
STAT3 signaling. Undifferentiated state mESCs expressed
LacdiNAc at a higher level than differentiated state cells.
Knockdown of b4GalNAc-T3 reduced LacdiNAc expression
and caused a decrease in LIF/STAT3 signaling that lessened
the rate of self-renewal of mESCs. A biochemical analysis
showed that LacdiNAc expression on LIF receptor (LIFR)
and gp130 was required for the stable localization of thereceptors with lipid raft/caveolar components, such as caveolin-1. This localization is required for transduction of a
sufﬁciently strong LIF/STAT3 signal. In primed state pluripotent stem cells, such as hiPSCs and mEpiSC-like cells
produced from mESCs, LacdiNAc expression on LIFR and
gp130 was extremely weak and the level of localization ofthese receptors on rafts/caveolae was also low. Furthermore, knockdown of b4GalNAc-T3 decreased LacdiNAc
expression and reduced the efﬁciency of reversion ofprimed state mEpiSC-like cells into naı̈ve state mESCs.
These ﬁndings show that the different LIF-responsivenessof naı̈ve state (mESCs) and primed state (mEpiSCs, hESCs,
and hiPSCs) cells is dependent on the expression of LacdiNAc on LIFR and gp130 and that this expression isrequired for the induction and maintenance of the naı̈ve
state. STEM CELLS 2011;29:641–650

Disclosure of potential conﬂicts of interest is found at the end of this article.

INTRODUCTION
Embryonic stem cells (ESCs) [1, 2] have considerable promisefor exploitation in biotechnology applications; in particular, thepluripotency of human ESCs (hESCs) will be valuable in cell
replacement therapies [3, 4]. One possible route for use of
ESCs in the development of regenerative medicine therapies
might be through exploitation of human induced pluripotent
stem cells (hiPSCs) [5]. In order to be able to exploit the rapid
culture methods and safety of hESCs and hiPSCs for therapeutic purposes, it will be important to elucidate the molecularmechanisms that control the self-renewal and differentiation of
ESCs. Many factors that control ESC pluripotency have now
been identiﬁed and it has been demonstrated that an appropriatebalance among several extrinsic signaling pathways is required
for the maintenance of pluripotency [6–8]. However, the underlying molecular mechanisms that determine the regulation ofthese extrinsic signaling pathways in ESCs remain unknown.

In recent years, several signaling pathways required for
self-renewal have been identiﬁed in mouse ESCs (mESCs)
and hESCs. One confounding factor, however, is that mouseand hESCs show differences in their responses to the extrinsic
signal factors required for self-renewal. For example, leukemia inhibitory factor (LIF)/signal transducer and activator oftranscription (STAT3) signaling contributes to the maintenance of self-renewal in mESCs but not hESCs [4, 9–13].
Bone morphogenic protein 4 (BMP4)/Smad signaling also
contributes to the maintenance of mESC self-renewal in synergy with LIF [14, 15]. By contrast, in hESCs, BMP signaling
induces differentiation [16, 17]. Wnt/b-catenin signaling contributes to the regulation of self-renewal in both mESCs andhESCs [18–21], whereas ﬁbroblast growth factor 2 (FGF2)
and activin/nodal signaling contribute only to the maintenanceof hESC self-renewal [22–24]. Thus, mESCs and hESCs
maintain their pluripotency using different signaling factors.
Recently, it was reported that primed state hESCs could be
induced to revert to a naı̈ve state similar to that of mESCs

N.S.: collection and/or assembly of data, data analysis and interpretation, manuscript writing. M.S.: collection and/or assembly of data,
data analysis and interpretation. K.H.: collection and/or assembly of data. K.U.: provision of study material or patients. S.N.: conception
and design, ﬁnancial support, manuscript writing, ﬁnal approval of manuscript.
Correspondence: Shoko Nishihara, Ph.D., Laboratory of Cell Biology, Department of Bioinfomatics, Faculty of Engineering, Soka
University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan. Telephone: 81-426-91-8140; Fax: 81-426-91-9315; e-mail: shoko@soka.
ac.jp Received November 15, 2010; accepted for publication January 27, 2011; ﬁrst published online in STEM CELLS EXPRESS February
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.615
8, 2011. V

STEM CELLS 2011;29:641–650 www.StemCells.com

LacdiNAc Is Essential for LIF/STAT3 Signaling

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and that these different states reﬂected differences in signaling
required for maintenance of self-renewal [25]. However, thekey factors that underlie the differences in signaling between
mESCs and hESCs have yet to be clariﬁed. One possibility isthat cell surface factors may be important for regulation of
extrinsic signaling. On this basis, we speculated that cell surface glycans might be a key factor in the different signaling
pathways for self-renewal in mESCs and hESCs.
Several types of glycan are expressed on the cell surface
and are present as components of glycoproteins, glycolipids,
and proteoglycans; these glycans thereby contribute signiﬁcantly to biologically important functions such as the regulation of signaling pathways [26]. It has recently been demonstrated that the cell surface glycan heparan sulfate (HS)contributes to self-renewal and differentiation of mESCs by
regulating BMP, Wnt, and FGF signaling [21, 27–29]. The
expression patterns of several other cell surface glycans havenow been described in undifferentiated and differentiatedmESCs and hESCs [30–34]. However, although these glycans
may be involved in the regulation of the signaling requiredfor ESC self-renewal, with the exception of HS, their functional roles have not been demonstrated.
To test our speculation that cell surface glycans might contribute to ESC self-renewal, we performed an RNA interference(RNAi) screen using short hairpin RNAs (shRNAs) that targeted
speciﬁc glycan-related genes in mESCs and sought to identify
cell surface glycans that were essential for ESC self-renewal. By
evaluation of alkaline phosphatase activity in RNAi-mediatedknockdown mESCs, we identiﬁed glycans essential for selfrenewal. One of the glycan sequences of interest is the LacdiNAc (GalNAcb1-4GlcNAc) motif, which is synthesized by
b4GalNAc-T3 [35]. LacdiNAc is frequently present on glycoproteins and glycolipids in invertebrates but is only present on alimited number of glycoproteins and glycolipids, such as glycoprotein hormones, in vertebrates [36, 37]. At present, the rolesof LacdiNAc in mammalian cells are not fully understood. Here,
we investigated the role of LacdiNAc and show that it contributes to the maintenance of the naı̈ve state in pluripotent cells by
regulating LIF/STAT3 signaling.

(http://sidirect.jp/esd/modules/modsiperfect/)’’ and are listed in supporting information Table 1. Transient transfection was performed
as described previously [21]. After selection of cells carrying
shRNA, they were harvested and analyzed as described below. Stable transfection was performed as described previously [29].
Mouse epiblast stem cell (mEpiSC)–like cells were induced
from mESCs as described previously [41]. In brief, mESCs were
harvested and 2  105 cells per dish were replated on 35 mm tissue
culture dishes precoated with 15 lg/ml Fibronectin (Sigma-Aldrich).
After 24 hours of culture, the medium was replaced with serum-free
medium consisting of DMEM/F12 supplemented with 48% Neurobasal (Invitrogen), 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 1 mM L-glutamine, 1% penicillin/streptomycin,
0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 5
mg/ml bovine serum albumin (BSA), with 20 ng/ml activin A(Wako) and 12 ng/ml FGF2. Thereafter, cells were passaged every
2–3 days using collagenase IV (Invitrogen). Cells that had been passaged three times were used for almost all experiments.
mEpiSC-like cells were induced to revert to mESCs as
described previously [25, 42]. In brief, the EpiSC-like cells wereseeded on inactivated MEFs as small clumps and cultured with serum-free medium containing 1,000 U/ml LIF, 1 lM PD0325901(Wako), 3 lM CHIR99021 (Wako), 10 lM forskolin (Stemgent,
San Diego, CA, www.stemgent.com), and 2 lM SB431542 (Wako).
A proliferation assay was performed as described previously [21].
Brieﬂy, transfected cells were replated in 96-well gelatin-coated plates
in mESC medium with LIF, and a solution of WST-8 from a Cell
Counting Kit-8 (Dojindo, Kumamoto, Japan, www.dojindo.co.jp) was
added after 0, 24, or 48 hours culture. WST-8 is reduced by cellulardehydrogenases to produce a soluble formazan product, which can bedetected colorimetrically at 450 nm. The intensity of staining is proportional to cell numbers. Manual cell counts were also performed to
conﬁrm the results of the proliferation assay.
Alkaline phosphatase staining was performed as described previously [21]. Brieﬂy, transfected cells were replated at clonal density in gelatin-coated 60 mm tissue culture dish in ESC medium
with LIF. After 5 days of culture, the cells were stained with 5bromo-4-chloro-3-indoxyl phosphate-nitro blue tetrazolium chloride(Nacalai Tesque, Kyoto, Japan, www.nacalai.co.jp), and alkaline
phosphatase positive colonies were counted under a microscope.

Fluorescence-Activated Cell Sorting (FACS) Analysis

MATERIALS AND METHODS
Cell Culture
R1 mESC lines [38] were maintained on mouse embryonic ﬁbroblasts (MEFs) inactivated with 10 lg/ml mitomycin C (Sigma,
Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich.com) in mESC
medium (Dulbecco’s modiﬁed Eagle’s medium [DMEM] supplemented with 15% fetal bovine serum [Hyclone Laboratories,
South Logan, UT, www.hyclone.com], 1% penicillin/streptomycin[Invitrogen, Invitrogen, Carlsbad, CA, www.invitrogen.com], 0.1
mM 2-mercaptoethanol [Invitrogen], 0.1 mM nonessential aminoacids [Invitrogen]) and 1,000 U/ml LIF [Chemicon International,
Temecula, CA, www.chemicon.com]. hiPSC clones (MRC-hiPS_Tic [JCRB1331]) [39] were maintained on inactivated MEFs in
hiPSC medium (DMEM/F12 supplemented with 20% KnockoutSerum Replacement [Invitrogen], 2 mM L-glutamine [Invitrogen],
1% penicillin/streptomycin, 0.1 mM 2-mercaptoethanol, 0.1 mM
nonessential amino acids) with 10 ng/ml FGF2 (Wako, Osaka, Japan, www.wako-chem.co.jp).
Knockdown of b4GalNAc-T3 was performed as described previously [21]. We generated shRNA expression vectors targetingb4GalNAc-T3 or enhanced green ﬂuorescent protein (EGFP) (negative control) by inserting the appropriate double-stranded DNAs
between the BamHI and HindIII sites of pSilencer 3.1-H1(Ambion, Austin, TX, www.ambion.com). The sequences used for
RNAi were designed, as described previously [40], using ‘‘siDirect

In general, trypsinization reduces the expression of some cell surface antigens. Therefore, to avoid this effect, cells were harvested
with accutase (Millipore, Billerica, MA, www.millipore.com) andthese dissociated single cells were incubated with ﬂuorescein isothiocyanate–conjugated Wistaria ﬂoribunda (WFA) (EY Laboratories, San Mateo, CA, www.eylabs.com) in FACS buffer (0.5%
BSA and 0.1% sodium azide in phosphate-buffered saline (PBS))
for 30 minutes on ice. After washing, cell sorting and analysis
were performed using a FACSAria Cell Sorter (Becton Dickinson, Franklin Lakes, NJ, www.bd.com).

Analysis of Proteins by Immunoblotting
For observation of cell signaling, the cell culture medium was
replaced with serum-free ESC medium without LIF for 4 hours andthe cells were stimulated for 20 minutes with 1,000 U/ml LIF or
10 ng/ml BMP4 (R&D Systems, Minneapolis, MN, www.rndsystems.com) or for 5 minutes with 10 ng/ml FGF4 (R&D Systems).
Cells were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150
mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 10 mM NaF, protease inhibitors). For immunoprecipitation, cells were lysed with lysis
buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100,
5 mM EDTA, protease inhibitors), and immunoprecipitation was
performed with appropriate antibody and Protein G Magnetic Beads(New England Biolabs, Ipswich, MA, www.neb.com).
Fractionation of lipid rafts/caveolae and nonrafts was performed as described previously [43]. Harvested cells were incubated with 0.5 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 150
mM NaCl, 1% Brij58 (Sigma-Aldrich), 1 mM Na3VO4, 10 mM

Sasaki, Shinomi, Hirano et al.

NaF, protease inhibitors) for 30 minutes on ice and homogenized
50 times with a tight Dounce homogenizer. The extract (1 ml)
was mixed with 85% sucrose solution (50 mM Tris-HCl pH 7.4
and 150 mM NaCl) to produce a 42.5% sucrose solution, transferred to a centrifuge tube (Beckman Coulter, Fullerton, CA,
www.beckmancoulter.com) and overlaid with 5 ml of 30% sucrose solution and 3 ml of 5% sucrose solution. The discontinuous sucrose gradients were centrifuged at 4 C for 16 hours in an
SW41 Ti rotor at 30,000 rpm. The gradient was divided into nine
fractions from the bottom to the top.
For extraction of WFA-binding proteins, a cell lysate prepared
as described above was incubated with WFA-agarose (Vector Laboratories, Burlingame, CA, www.vectorlabs.com) at 4 C, washedwith 0.1% Triton X-100 in tris-buffered saline, and WFA-binding
proteins were extracted with 0.2 M lactose (Sigma-Aldrich).
SDS–polyacrylamide gel electrophoresis (PAGE) samples prepared as described above were separated by the appropriate percentage SDS-PAGE and transferred onto polyvinylidene ﬂuoride
membranes (Millipore). After blocking, the membranes were incubated with the following primary antibodies: anti-STAT3 (Becton
Dickinson), anti-phospho-STAT3 (Tyr705; Becton Dickinson),anti–extracellular signal-regulated kinase 1/2 (anti-ERK1/2) (Cell
Signaling Technology, Danvers, MA, www.cellsignal.com), antiphosphorylated ERK1/2 (Thr183/Thr185; Cell Signaling Technology),
anti-phospho-Smad1 (Ser463/Ser465; Cell Signaling Technology),
anti-b-actin (Sigma-Aldrich), anti-Flotillin-1 (Becton Dickinson), antiTransferrin receptor (Zymed, San Francisco, CA, www.zymed.com),anti-LIF receptor (LIFR) (C-19; Santa Cruz Biotechnology, Santa
Cruz, CA, www.scbt.com), anti-gp130 (M-20; Santa Cruz Biotechnology), anti-caveolin-1 (7C8; Santa Cruz Biotechnology), anti-Oct3/4(C-10; Santa Cruz Biotechnology), anti-Nanog (ReproCELL, Yokohama, Japan, www.reprocell.com), or anti-Sox2 (R&D Systems). The
membranes were then incubated with the appropriate peroxidase-conjugated secondary antibodies (Cell Signaling Technology), washed,
and developed with ECL Plus reagents (GE Healthcare, Waukesha,
WI, www.gehealthcare.com). For lectin blot analysis, the membranes
were incubated with peroxidase-conjugated WFA (EY Laboratories),
washed, and developed with ECL Plus reagents.
Cell surface expression of LIFR and gp130 was examined bybiotinylation of cell surface proteins followed by immunoprecipitation. Biotinylation of cell surface proteins was performed using
EZ-Link sulfosuccinimidyl-6-(biotin-amido) hexanoate (PIERCE,
Rockford, IL, www.piercenet.com) in accordance with the manufacturer’s protocol. Then, biotinylated cell surface proteins were
immunoprecipitated with anti-LIFR antibody or anti-gp130 antibody as described above. Immunoprecipitated proteins were subjected to Western blot analysis using peroxidase-conjugated streptavidin (Wako). Similar to the FACS analysis above, we did not
use trypsinization to harvest the cells used for analyzing cell surface proteins. Instead, the cells were harvested using 0.05%
EDTA and a cell scraper.

Immunostaining of Nonpermeabilized Cells
mESCs, mEpiSC-like cells, or hiPSCs were ﬁxed with 2% paraformaldehyde, washed, and subsequently blocked with PBS containing 3% BSA and 1% normal goat serum. After washing, nonpermeabilized cells were double-stained with an anti-caveolin-1
antibody and an anti-LIFR or anti-gp130 antibody. After washing,cells were stained with Alexa488-conjugated secondary antibody(Invitrogen) and Alexa594-conjugated secondary antibody (Invitrogen). Immunoﬂuorescence images were taken using an LSM5Pascal
confocal laser scanning microscope (Carl Zeiss, Thornwood, NY,
www.zeiss.com) with 63x/1.3 objective at room temperature.

Real-Time PCR
Real-time PCR was performed as described previously [21]. For
some genes, FastStart Universal SYBR Green Master (Roche,
Basel, Switzerland, www.roche-applied-science.com) was used.
Primer sets and probes for real-time PCR are listed in supporting
information Tables 2 and 3, respectively.

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643

RESULTS
LacdiNAc Contributes to Self-Renewal of mESCs
To identify cell surface glycans involving in self-renewal of
mESCs, we performed an RNAi screen using shRNAs thattargeted various glycan-related genes in mESCs. By evaluating
alkaline phosphatase activity in glycan-related gene knockdown
mESCs, LacdiNAc, which is synthesized by b4GalNAc-T3 [35],
was identiﬁed as a candidate cell surface glycan required for
self-renewal of mESCs. We designed two constructs that
expressed different shRNAs targeting b4GalNAc-T3 (b4GalNAcT3-1 and b4GalNAc-T3-2), as described previously [40], andadditionally constructs that expressed shRNAs targeting EGFP
as negative controls. We describe mESCs that have been transfected with EGFP shRNA expression plasmids as ‘‘control cells’’
throughout this article. b4GalNAc-T3-knockdown (KD) cells
showed an approximately 70% reduction in b4GalNAc-T3mRNA when compared with control cells (Fig. 1A). FACS analysis using WFA lectin, which recognizes LacdiNAc [44],showed that cell surface expression of LacdiNAc was also
reduced in b4GalNAc-T3-KD cells (Fig. 1B).
The proportion of alkaline phosphatase positive colonies
in cultures of b4GalNAc-T3-KD cells after replating was markedly decreased when compared with those of control cells evenin the presence of LIF and serum, demonstrating that the b4GalNAc-T3-KD cells had lost this stem cell trait (Fig. 1C). Furthermore, after 4 days of culture, even at normal density, the expression of Oct3/4, Nanog, and Sox2 markers of the undifferentiatedstate was signiﬁcantly decreased in b4GalNAc-T3-KD cells
when compared with control cells at both the mRNA and protein levels (Fig. 1D, 1E); thus, the ability for self-renewal inb4GalNAc-T3-KD cells had been abolished. These results indicate that LacdiNAc contributes to self-renewal of mESCs. As
shown in Figure 1F, cell surface expression of LacdiNAc wasdecreased following LIF withdrawal, suggesting that LacdiNAc
plays functionally important roles in undifferentiated mESCs. In
addition, the rate of proliferation of b4GalNAc-T3-KD cells
decreased signiﬁcantly when compared with that of control cells(Fig. 1G; supporting information Fig. S1). Taken together, theseresults demonstrate that LacdiNAc functions in undifferentiatedmESCs and is required for their self-renewal and proliferation.

LacdiNAc Regulates LIF/STAT3 Signaling
Next, we examined whether LacdiNAc is involved in the
extrinsic signaling pathways required for maintenance of selfrenewal. A Western blot analysis showed that LIF-induced phosphorylation of STAT3 occurred at a substantially reduced rate inb4GalNAc-T3-KD cells when compared with control cells, demonstrating that LacdiNAc has a role in LIF/STAT3 signaling(Fig. 2A). In contrast, we observed similar increases in the levelof phosphorylation of Smad1 or ERK in control and b4GalNAcT3-KD cells following exposure to BMP4 or FGF4 (supporting
information Fig. S2), indicating that LacdiNAc does not participate in BMP4/Smad or FGF4/ERK signaling.
We then addressed the question of how LacdiNAc might
regulate LIF/STAT3 signaling. Signal transduction via the
LIF/STAT3 pathway is mediated by the binding of LIF to
LIFR, and heterodimerization of LIFR and gp130 is subsequently induced [45]. This heterodimerization triggers the activation of associated Janus kinase tyrosine kinases followed byphosphorylation of gp130 and results in the activation of
STAT3. Here, we ﬁrst examined the levels of LIFR and gp130
on the cell surface by a Western blot analysis. LIFR and gp130
located on the cell surface were biotinylated and then immunoprecipitated; the amounts of biotinylated LIFR or biotinylated

644

LacdiNAc Is Essential for LIF/STAT3 Signaling

Figure 1. Self-renewal and proliferation is reduced in b4GalNAc-T3-KD cells. (A): Real-time PCR analysis of cells 2 days after transfection.
Relative amounts of b4GalNAc-T3 mRNA were calculated after normalization against b-actin mRNA. The results are shown after normalization
against the value obtained in control cells (value ¼ 1). The values shown are the means 6 SD of three independent experiments. (B): FACS
analysis of cells 2 days after transfection using FITC-Wistaria ﬂoribunda. Three independent experiments were performed and representative
results are shown. The mean intensities of FITC staining were 23,947, 17,400, and 18,204 in the control, b4GalNAc-T3-1-KD, and b4GalNAcT3-2-KD cells, respectively. (C): Alkaline phosphatase staining. The ratio of alkaline phosphatase positive colonies is shown after normalization
to the ratio obtained in control cells (value ¼ 1). The values shown are the means 6 SD from three independent experiments and signiﬁcant values are indicated, *, p < .01, in comparison to the control. (D): Real-time PCR analysis of Oct3/4, Nanog, and Sox2 markers of the undifferentiated state
at 4 days after transfection. The results are shown after normalization against the values obtained for control cells (value ¼ 1). The values shown are themeans 6 SD from two independent experiments. (E): Western blot analysis of Oct3/4, Nanog, and Sox2 at 4 days after transfection. (F): FACS analysis
of cells 4 days after culture with or without leukemia inhibitory factor (LIF). Three independent experiments were performed and representative results areshown. The mean intensities of FITC staining were 22,954 and 13,315 in LIFþ and LIF cells, respectively. (G): Proliferation assay using cell counting
kit-8. The values shown are the means 6 SD from three independent experiments. Abbreviations: KD, knockdown; LIF, leukemia inhibitory factor.

gp130 were determined by a Western blot analysis. As shownin Figure 2B, there were no signiﬁcant differences in cell surface expression of LIFR and gp130 between control andb4GalNAc-T3-KD cells. This result indicates that the reductionin LIF/STAT3 signaling in b4GalNAc-T3-KD cells was notcaused by variation in the relative expression levels of LIFR

and gp130 on the cell surface and that LacdiNAc did not affectthe levels of these receptors on the cell surface.
We then investigated whether LacdiNAc is required forthe heterodimerization of LIFR and gp130. A Western blot
analysis showed that gp130 on control cells could be detected
in immunoprecipitates using an anti-LIFR antibody and,

Sasaki, Shinomi, Hirano et al.

645

Figure 2. Leukemia inhibitory factor (LIF)/STAT3 signaling is reduced in b4GalNAc-T3-KD cells. (A): Western blot analysis of mouse embryonic stem cells stimulated with LIF 2 days after transfection. The histograms show mean densitometric readings 6 SD of the phosphorylated protein/loading controls. Values were obtained from duplicate measurements of three independent experiments and signiﬁcant values are indicated,*, p < .01, in comparison to the stimulated control. (B): Western blot analysis of cell surface LIF receptor (LIFR) and gp130. Two days after
transfection, cells were biotinylated for detection of cell surface LIFR and gp130. After lysis, biotinylated cell surface proteins were immunoprecipitated with anti-LIFR or anti-gp130 antibody. Immunoprecipitates were detected by horseradish peroxidase–streptavidin (ﬁrst and fourth panels). Second and ﬁfth panels represent total expression levels of LIFR and gp130. Negative control staining without the primary antibody did notshow nonspeciﬁc bands of the size expected of either LIFR or gp130 (third and sixth panels). (C): Western blot analysis of heterodimerization of
LIFR and gp130 in response to LIF 2 days after transfection. Top panel shows Western blot analysis of gp130 coimmunoprecipitated with LIFR.
Middle top panel shows amounts of immunoprecipitated LIFR, and middle lower and lower panels show total expression levels of LIFR and
gp130. (D): Western blot analysis of LIFR and gp130 in raft (Fr.3–5) and nonraft (Fr.7–9) fractions 2 days after transfection. Abbreviations: Ab,
antibody; IB, immunoblot; IP, immunoprecipitate; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory factor receptor; ND, not detected;
p-STAT3, phospho-STAT3; STAT3, signal transducer and activator of transcription 3.

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LacdiNAc Is Essential for LIF/STAT3 Signaling

moreover, that the amount of gp130 varied with the concentration of LIF used to stimulate the cells. This observation
conﬁrms that gp130 forms a heterodimer with LIFR in
response to LIF (Fig. 2C; top panel; ﬁrst and fourth lanes). In
contrast, in b4GalNAc-T3-KD cells, the levels of gp130 present in immunoprecipitates with the anti-LIFR antibody weresigniﬁcantly reduced when compared with control cells (Fig.
2C; top panel; ﬁfth and sixth lanes), although the total
amounts of LIFR and gp130 did not change (Fig. 2C; middle
lower and lower panels). This result clearly indicates that theability of LIFR and gp130 to heterodimerize in response to
LIF was reduced in b4GalNAc-T3-KD cells.
A recent study demonstrated that LIFR and gp130 are localized in lipid rafts/caveolae and that this localization is requiredfor LIF/STAT3 signaling in mESCs [46]. Therefore, we examined the localization of LIFR and gp130 by isolating lipid rafts.
The separation of lipid rafts and nonrafts was conﬁrmed byWestern blot analysis using an anti-Flotillin-1 antibody for theraft fractions (fractions 3–5) and an antitransferrin receptor antibody for the nonraft fractions (fractions 7–9) (supporting information Fig. S3). As shown in Figure 2D, the relative levels of
LIFR and gp130 in raft fractions (fractions 3–5) were considerably reduced in b4GalNAc-T3-KD cells when compared with
control cells. This result indicates that LIFR and gp130 weredispersed from lipid rafts/caveolae in b4GalNAc-T3-KD cells.
Overall, the ﬁndings in these various experiments demonstratethat LacdiNAc is involved in LIF/STAT3 signaling in mESCs
through regulating the localization of LIFR and gp130 to rafts/
caveolae and through controlling their heterodimerization.

LacdiNAc on LIFR and Gp130 Is Required for
Raft/Caveolar Localization
The results described above indicate that LacdiNAc sequenceson LIFR and gp130 inﬂuence their localization to rafts/caveolae. However, to date, the occurrence of this motif on LIFR
and gp130 has not been demonstrated. We investigatedwhether LacdiNAc sequences are present on LIFR and gp130
using WFA-binding proteins (LacdiNAc-expressing proteins)
puriﬁed from mESCs using WFA-agarose. As shown in Figure 3A, both LIFR and gp130 were observed in the fractions
eluted using the WFA-binding proteins, indicating that LacdiNAc is expressed on LIFR and gp130. We also found that theamounts of LIFR and gp130 that could be puriﬁed fromb4GalNAc-T3-KD cells were reduced when compared with
control cells (Fig. 3B) indicating that expression of LacdiNAc
on LIFR and gp130 was reduced in b4GalNAc-T3-KD cells.
\The localization of signal receptors to lipid rafts is believed
to be regulated by the association of signal receptors with components of the rafts, such as caveolin-1 and glycolipids. We
found that both LIFR and gp130 colocalized with caveolin-1 on
the surfaces of mESCs (supporting information Fig. S5). Thus,
we carried out biochemical analysis to determine whether LIFR
and gp130 interact speciﬁcally with particular components of therafts/caveolae, such as caveolin-1, and also whether this interaction is dependent on LacdiNAc expression. A Western blot analysis of immunoprecipitates obtained using the anti-caveolin-1
antibody identiﬁed comparatively low amounts of LIFR and
gp130 from b4GalNAc-T3-KD cells when compared with control cells, demonstrating that interaction of caveolin-1 with LIFR
and gp130 was reduced in b4GalNAc-T3-KD cells (Fig. 3C).
This result indicates that caveolin-1 binds to LIFR and gp130 byrecognizing LacdiNAc sequences on these receptors.
On the basis of these results, we propose that LacdiNAc
sequences on LIFR and gp130 are required for the localization of these receptors to the rafts/caveolae by interaction
with components such as caveolin-1.

Figure 3. LacdiNAc on leukemia inhibitory factor receptor (LIFR)
and gp130 contributes to interaction of these receptors with caveolin1. (A): Western blot analysis of Wistaria ﬂoribunda (WFA)-binding
proteins. Immunoblots with anti-LIFR or anti-gp130 antibody or lectin blot with horseradish peroxidase–WFA are shown. Input, ﬂowthrough, wash, and eluted fractions were prepared from mouse embryonic stem cells as described in the Materials and Methods section.
(B): Western blot analysis of WFA-binding LIFR or gp130 2 days after transfection. Immunoblot with anti-LIFR or anti-gp130 antibody
are shown. Eluted fractions from WFA-binding proteins were prepared as described in the Materials and Methods section. (C): Western blot analysis of interaction between caveolin-1 and LIFR or
gp130 2 days after transfection. Immunoblot of LIFR or gp130 coimmunoprecipitated with caveolin-1 are shown. Abbreviations: Cav1,
caveolin-1; IB, immunoblot; IP, immunoprecipitate; LIFR, leukemia
inhibitory factor receptor; WFA, Wistaria ﬂoribunda.

LacdiNAc on LIFR and gp130 Is a Key Factor for
LIF/STAT3 Signaling in Naı̈ve State Pluripotent
Stem Cells
It is known that although LIF/STAT3 signaling maintains
self-renewal of mESCs, it does not function in either
mEpiSCs or hESCs [4, 13, 47, 48]. The underlying molecular mechanisms of this differential response to LIF have yet
to be clariﬁed. We examined this question by ﬁrst comparing
the levels of LacdiNAc on mESCs and mEpiSC-like cells(produced from mESCs in EpiSC culture conditions as previously reported [41]). mEpiSC-like cells show characteristicfeatures of mEpiSCs such as a monolayer morphology andmarker gene expression (supporting information Fig. S4).
We found that cell surface expression of LacdiNAc was
lower in mEpiSC-like cells than in mESCs (Fig. 4A). Moreover, expression of b4GalNAc-T3 was also lower in
mEpiSC-like cells (Fig. 4B). Similar to mEpiSCs andhESCs, the mEpiSC-like cells showed a very weak response
to LIF (Fig. 4C). The presence of LacdiNAc sequences on
LIFR and gp130 is required for LIF/STAT3 signaling inSasaki, Shinomi, Hirano et al.

647

Figure 4. The presence of LacdiNAc on leukemia inhibitory factor receptor (LIFR) and gp130 is required for LIF/STAT3 signaling in naı̈ve
state. (A): FACS analysis of LacdiNAc on mouse embryonic stem cell (mESCs) and mouse epiblast stem cell (mEpiSC)-like cells using FITCWistaria ﬂoribunda (WFA). Mean FITC staining intensities of 20,959 and 13,476 were obtained for mESCs and mEpiSC-like cells, respectively.
(B): Real-time PCR analysis of b4GalNAc-T3 in mESCs and mEpiSC-like cells. Relative amounts of b4GalNAc-T3 were calculated after normalization to b-actin mRNA and shown after normalization against the value obtained in mESCs (value ¼ 1). The values shown are the means 6
SD of duplicate measurements. (C): Western blot analysis of mESCs and mEpiSC-like cells stimulated with LIF. (D): Western blot analysis of
WFA-binding LIFR or gp130 in mESCs, mEpiSC-like cells, and human induced pluripotent stem cells (hiPSCs). Immunoblot with anti-LIFR,
anti-gp130, or anti-b-actin antibody is shown. Eluted fractions from WFA-binding proteins were prepared as described in the Materials and Methods section. (E): Western blot analysis of LIFR and gp130 in raft (Fr.3–5) and nonraft (Fr.7–9) fractions in mESCs, mEpiSC-like cells, andhiPSCs. (F): Reversion of b4GalNAc-T3-KD EpiSC-like cells. Left panel, macroscopic view of alkaline phosphatase-stained dishes 4 days after
reversion is shown. Right panel, relative number of alkaline phosphatase-positive colonies after normalization against the value obtained in control cells (value ¼ 1) is shown. The values shown are the means 6 SD from three independent experiments and signiﬁcant values are indicated,*, p < .01, in comparison to the control. Abbreviations: hiPSC, human induced pluripotent stem cell; IB, immunoblot; LIF, leukemia inhibitory
factor; LIFR, leukemia inhibitory factor receptor; mESC, mouse embryonic stem cell; mEpiSC, mouse epiblast stem cell; p-STAT3, phosphoSTAT3; STAT3, signal transducer and activator of transcription 3; WFA, Wistaria ﬂoribunda.

mESCs (Figs. 2 and 3 and above description). We postulated
that expression of LacdiNAc on these receptors was reduced
in EpiSC-like cells. Although the total levels of LIFR and
gp130 expression were lower in mEpiSC-like cells thanmESCs, the rate of LacdiNAc expression on LIFR and
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gp130 was absolutely lower in mEpiSC-like cells thanmESCs (Fig. 4D). This result indicates that expression of
LacdiNAc sequences on LIFR and gp130 was reduced in
EpiSC-like cells. Furthermore, the levels of LacdiNAc on
LIFR and gp130 were also extremely low in hiPSCs (Fig.

LacdiNAc Is Essential for LIF/STAT3 Signaling

648

4D). Next, we compared the localization of LIFR and gp130
on rafts/caveolae in mEpiSC-like cells, hiPSCs, and mESCs.
Expression of LIFR and gp130 in the raft fractions was low
in both mEpiSC-like cells and hiPSCs when compared tomESCs, indicating that raft/caveolar localization of LIFR
and gp130 was weak in both mEpiSC-like cells and hiPSCs(Fig. 4E). In addition, immunocytostaining showed that both
LIFR and gp130 colocalized poorly with caveolin-1 in
mEpiSC-like cells and hiPSCs (supporting information Fig.
S5). From these results, we propose the following molecularmechanism for the weak response to LIF in primed state pluripotent stem cells, such as EpiSCs and hESCs including
hiPSCs: LIFR and gp130 do not localize in rafts/caveolae
because of the low level of LacdiNAc, which results in the
weak transduction of LIF/STAT3 signaling.
A recent study reported that LIF/STAT3 signaling contributes to reversion from the primed state to the naı̈ve state [49–51]. Therefore, we investigated whether the regulation of LIF/
STAT3 signaling by LacdiNAc expression contributed toreversion. We used mEpiSC-like cells produced from stableb4GalNAc-T3-KD cells. b4GalNAc-T3-KD cells produced
mEpiSC-like cells similar to control cells in morphology andmarker gene expression (supporting information Fig. S6). We
found that the rate of reversion in mEpiSC-like cells derived
from b4GalNAc-T3-KD cells was markedly lower than incontrol cells (Fig. 4F). This result indicates that upregulationof LacdiNAc is required for reversion from the primed state
to the naı̈ve state. We conclude that one factor in the differential response of naı̈ve and primed state pluripotent cells to
LIF is the levels of LacdiNAc sequences on LIFR and gp130
and that expression of LacdiNAc is required for the induction
and maintenance of the naı̈ve state in pluripotent stem cells.

DISCUSSION
The extrinsic signals required for self-renewal differ between
mESCs and hESCs. However, the molecular mechanism that
underlies this differential behavior has not yet been clariﬁed.
In recent years, pluripotent stem cells at different developmental stages have been identiﬁed, such as EpiSCs and FGF/
activin/Bio-stem cells (FAB-SCs) in the mouse [46, 47, 52].
EpiSCs are derived from the postimplantation epiblast andhave similar features to hESCs. In particular, both cell types
are maintained by the same extrinsic signals, namely, activin/nodal and FGF2 signaling. Recent studies have demonstrated
that EpiSCs, which are more advanced developmentally thanmESCs, can be induced to revert to mESC-like cells [41, 42,
49–51]. FAB-SCs are generated from the preimplantation epiblast under deﬁned culture conditions, including the presenceof FGF2 and activin, and they can be induced to revert to themESC-like state. The relative developmental stage of FABSCs is uncertain but they are thought to be more developmentally advanced than mESCs. Thus, the information obtained
from several studies, using various mouse stem cells derived
from developmentally different stages, indicates that hESCs
are at a developmentally later stage that corresponds to thepostimplantation epiblast (primed state). By contrast, mESCs
are derived from ICM and are maintained in the naı̈ve state.
Recent reports indicate that hESCs (primed state) can be
induced to revert to mESC-like cells (naı̈ve state) under the
speciﬁc culture conditions that are required for maintenanceof mESCs; after reversion, these naı̈ve hESCs can be maintained under culture conditions containing LIF [25, 53]. Thus,
these reports have reinforced the view that hESCs and mESCs
are at developmentally different stages. This difference in de-Figure 5. Schematic representation of leukemia inhibitory factor(LIF)/STAT3 signaling regulated by LacdiNAc in naı̈ve and primed
state cells. In the naı̈ve state, LacdiNAc expressed on LIF receptor(LIFR) and gp130 contributes to the interaction with raft/caveolar
components, such as caveolin-1, and this interaction stabilizes theraft/caveolar localization of LIFR and gp130 and results in strongtransduction of the LIF/STAT3 signal. Thus, naı̈ve state mouse embryonic stem cells and presumably converted cells can be maintained in
an undifferentiated state by LIF/STAT3 signaling. In the primed state,
LIFR and gp130 are weakly localized to raft/caveolae due to the lack of
LacdiNAc on the receptors, which results in weak transduction of the
LIF/STAT3 signal. Therefore, self-renewal of mouse epiblast stem cells,
human induced pluripotent stem cells, and presumably human ESCs cannot be maintained by LIF/STAT3 signaling. Abbreviations: Cav1, caveolin-1; hiPSC, human induced pluripotent stem cell; LIF, leukemia
inhibitory factor; LIFR, leukemia inhibitory factor receptor; mESC,
mouse embryonic stem cell; mEpiSC, mouse epiblast stem cell.

velopmental stage between hESCs and mESCs is also
reﬂected in their different responses to extrinsic signals. In
particular, while LIF/STAT3 signaling contributes to themaintenance of self-renewal in mESCs, it does not do so for
hESCs [4, 13]. However, the factors responsible for this variation in LIF-dependency have yet to be identiﬁed. In thisstudy, we have shown that expression of LacdiNAc on LIFR
and gp130 is required for their localization to rafts/caveolae;
this expression is required for strong transduction of LIF/
STAT3 signaling in naı̈ve state cells, and the low expressionof primed states cells is associated with weak signal transduction. To the best of our knowledge, this is the ﬁrst demonstration that differential expression of LacdiNAc on LIFR and
gp130 in naı̈ve and primed state cells underlies the different
responses of mESCs (naı̈ve state) and hESCs, including
mouse EpiSCs (primed state), to LIF/STAT3 signaling.

Sasaki, Shinomi, Hirano et al.

The molecular mechanisms of signal transduction mediated by interleukin-6 and related cytokines, such as LIF, arewell-deﬁned [45]. More recently, the contribution of glycans
to LIF/STAT3 signaling has been reported in neural stem
cells, in which N-glycans contribute to the heterodimerizationof LIFR and gp130 [54]. However, the molecular mechanisms
by which N-glycans mediate heterodimerization have not beenclariﬁed. In this study, we demonstrated that reduction of
LacdiNAc, which can be present on N-glycans or O-glycans,resulted in the inhibition of heterodimerization and a decreasein LIF/STAT3 signaling. Furthermore, we found that a reduction in LacdiNAc sequences induced defective localization of
LIFR and gp130 to rafts/caveolae. The rafts/caveolae provide
an effective platform for signal transduction by enrichment ofseveral signal receptors, thereby forming an efﬁcient complexof receptors and other signal-related factors. Therefore, we
propose that deﬁcient heterodimerization of LIFR and gp130
as a consequence of a reduction in LacdiNAc is dependent on a
reduction in the raft/caveolar localization of these receptors.
Nevertheless, it is also possible that LacdiNAc sequences
directly contribute to the interaction between LIFR and gp130;
further study will be required to examine this possibility.
It was recently reported that LIFR and gp130 localizationin rafts/caveolae in mESCs is required for LIF/STAT3 signaling [46]. This study found that knockdown of caveolin-1
did not affect raft/caveolar localization of LIFR and gp130.
Here, we demonstrated that the interaction of LIFR and
gp130 with caveolin-1 was mediated by LacdiNAc. Therefore, we propose that LacdiNAc mediates the interaction of
LIFR and gp130 not only with caveolin-1 but also with otherraft/caveolar components, such as polymerase I transcript
release factor/cavin-1, caveolin-2, and glycosylphosphatidylinositol-anchored proteins, to stabilize the localization ofthese receptors. We shall examine this proposal in futurestudies. To date, it has been demonstrated that the bindingof some raft/caveolar associated proteins to caveolin-1 ismediated by the caveolin binding motif [55]. We found that
LIFR and gp130 have the caveolin binding motifs YGTVVFAGY (amino acids 318–326) and FTFTTPKF (amino acids
604–611), respectively. Both motifs contain threonine residues that might possibly be modiﬁed by O-linked glycosylation. It is possible that the O-glycans modiﬁed on the threonine residues of LIFR or gp130 have LacdiNAc sequences.
Thus, we suggest that LacdiNAc modiﬁcation on the caveolin binding motif might affect the interaction between caveolin-1 and LIFR or gp130; further investigation will beneeded to conﬁrm this possibility.
It has been reported that LIF/STAT3 signaling contributes
to the reversion of primed state EpiSCs to naı̈ve state mESCs[49, 50]. Additionally, a recent study showed that weak transduction of the LIF/STAT3 signal is a possible barrier to thisreversion [51]. Here, our ﬁndings indicated that expression of
LacdiNAc on LIFR and gp130 was required for the reversion
process. Furthermore, it has previously been shown that naı̈ve
state hESCs and metastable nonobese diabetic-iPSCs can be
induced under deﬁned culture conditions [25]; these cells can

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ACKNOWLEDGMENTS
This research was supported in part by Ministry of Education, Culture, Sports, Science and Technology (MEXT); the Grant-in-Aid
for Scientiﬁc Research (B), grant 20370051 for the years 2008–2010 (to S.N.); and the Matching Fund for Private Universities
from MEXT, grant S0901015 for the years 2009–2014.

DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conﬂicts of interest.

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