EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
E-Cadherin-Mediated Cell–Cell Contact Is Critical for Induced
Pluripotent Stem Cell Generation
TAOTAO CHEN,a DETIAN YUAN,a BIN WEI,a JING JIANG,a JIUHONG KANG,a,b KUN LING,c
YIJUN GU,a JINSONG LI,a LEI XIAO,a GANG PEIa,b
a
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai, China; bShanghai Key Laboratory of Signaling and
Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China; cDepartment of
Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Rochester, Minnesota, USA
Key Words. Cell adhesion molecules • Reprograming • Induced pluripotent stem • Embryonic stem cells
ABSTRACT
The low efficiency of reprogramming and genomic integration of virus vectors obscure the potential application
of induced pluripotent stem (iPS) cells; therefore, identification of chemicals and cooperative factors that may
improve the generation of iPS cells will be of great value.
Moreover, the cellular mechanisms that limit the reprogramming efficiency need to be investigated. Through
screening a chemical library, we found that two chemicals
reported to upregulate E-cadherin considerably increase
the reprogramming efficiency. Further study of the process indicated that E-cadherin is upregulated during
reprogramming and the established iPS cells possess Ecadherin-mediated cell–cell contact, morphologically indis-
tinguishable from embryonic stem (ES) cells. Our experiments also demonstrate that overexpression of E-cadherin
significantly enhances reprogramming efficiency, whereas
knockdown of endogenous E-cadherin reduces the efficiency. Consistently, abrogation of cell–cell contact by the
inhibitory peptide or the neutralizing antibody against the
extracellular domain of E-cadherin compromises iPS cell
generation. Further mechanistic study reveals that adhesive binding activity of E-cadherin is required. Our
results highlight the critical role of E-cadherin-mediated
cell–cell contact in reprogramming and suggest new
routes for more efficient iPS cell generation. STEM CELLS
2010;28:1315–1325
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Induced pluripotent stem (iPS) cells can be generated by
direct reprogramming of somatic cells through ectopic
expression of defined transcription factors, classically a
combination of four factors, Oct3/4, Sox2, c-Myc, and Klf4
[1–3]. iPS cells represent an intriguing new source for
patient-specific pluripotent stem cells [4–6]; therefore, many
efforts have been taken to make iPS cells more amenable
for therapeutic application. To improve the iPS generation
efficiency and avoid the oncogenic effect of viral vectors,
reduced number of factors as well as small molecules was
used to optimize the iPS generation technology [7, 8].
However, the reprogramming process is still highly inefficient [9]. A better understanding of the mechanisms underlying the reprogramming process might help us identify
other pathways or cooperative factors to improve the
reprogramming efficiency.
Small chemicals that can enhance the generation of iPS
cells are of great value because potentially they may replace
the oncogenic factors used in the current reprogramming protocol. So far, several chemicals were reported to improve
reprogramming efficiency. Among them, some were chromatin-modifying agents, such as valproic acid (VPA), 5-aza-20 deoxycitydine (AZA), and BIX-01294 [7, 10, 11]; others were
mainly inhibitors or activators of certain signal transduction
pathways (including mitogen activated protein kinase
(MAPK), glycogen synthase kinase 3 beta (GSK3b), and
transforming growth factor beta (TGFb) pathways) [10, 12–
14]. Through a high throughput screening, we found two
chemicals that reported to upregulate E-cadherin expression
had positive effect on the generation of iPS cells. E-cadherin
belongs to a large family of transmembrane proteins that
mediate specific cell–cell adhesion in a Ca2þ-dependent manner [15–17]. Each type of cells expresses certain subsets of
cadherins, which is essential to establish the basis of cell-specific identity [18]. In ES cells, it is currently understood that
Author contributions: T.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript
writing; D.Y.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; B.W.:
data analysis and interpretation, manuscript writing; J.J.: collection and/or assembly of data, data analysis and interpretation; J.K.: data
analysis and interpretation; K.L.: data analysis and interpretation; Y.G.: collection and/or assembly of data; J.L.: data analysis and
interpretation; L.X.: data analysis and interpretation; G.P.: conception and design, data analysis and interpretation, manuscript writing,
financial and administrative support, and final approval of manuscript. T.C. and D.Y. contributed equally to this work.
Correspondence: Gang Pei, Ph.D., Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Tel: þ86-21-54921373; fax: þ86-21-54921372; e-mail: gpei@
sibs.ac.cn Received March 23, 2010; accepted for publication May 21, 2010; first published online in STEM CELLS EXPRESS June 2,
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.456
2010. V
STEM CELLS 2010;28:1315–1325 www.StemCells.com
E-Cadherin is Critical for the Genaration of iPS Cells
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E-cadherin is the main cadherin molecule and regulates cell
growth not only through its adhesive binding activity but also
through its signal transducing activity [19–21]. Our previous
ChIP-chip studies investigated the genome-wide promoter occupancy of the ‘‘Yamanaka factors’’ and identified 16 signaling pathways regulated by these factors in iPS cells [22].
Interestingly, among these, there are four pathways, adhesion
junction, gap junction, focal adhesion, and tight junction,
which regulate complicated intercellular cross talk. Taken together, these results lead us to further investigate the role of
E-cadherin molecule in the reprogramming process.
Here we report that two small molecules with reported
activity to upregulate E-cadherin expression can increase the
reprogramming efficiency. We find that E-cadherin expression
is upregulated in the early stage of reprogramming and the
cell–cell contact in iPS cells is mainly mediated by E-cadherin. Enhanced iPS cell generation can be achieved by overexpression of E-cadherin, whereas knockdown of endogenous
E-cadherin or abrogation of cell–cell contact by E-cadherin
inhibition results in reduced reprogramming efficiency. Further mechanistic study demonstrates that the adhesive binding
activity is essential for the reprogramming process.
MATERIALS
AND
METHODS
Derivation of mouse embryonic fibroblast (MEF) Cells
MEF cells were isolated from e13.5 embryos heterozygous
for the Oct4::GFP transgenic allele, as previously described
[7]. Gonads and internal organs were removed when the
embryos were processed for isolation of MEF cells. Isolated
MEF cells in early passages (up to passage 4) were used for
further experiments.
Chemical Screening
MEF cells at passage 2 or 3 were seeded onto six-well plates at
a density of 200,000 per well in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FBS and transduced
with pMXs based retroviruses carrying the four factors. Transduced cells were split onto feeders in 96-well plates at a density
of 5,000 per well in KO-DMEM (Invitrogen, Carlsbad, CA,
http://www.invitrogen.com) supplemented with 1000 U/ml leukemia inhibitory factor (LIF) and 15% KSR (Invitrogen) on day
3 and 1 lM of each chemical was added to the culture on day
4. The medium was changed every other day. VPA (1 mM),
was added from day 4 to 9 and served as a positive control.
The other chemicals were added till the end of the experiment.
On day 18, the cells were fixed and GFP-positive colonies were
counted under a fluorescent microscope (Zeiss Axio Observer
Z1). The compounds we used in the initial screening were provided by Chinese National Compound Resource Center. The
library contains approximately 500 natural compounds and
>1,500 synthetic compounds with known biological activities.
Construction of Retroviral Vectors
For overexpression of the E-cadherin protein, coding sequence
of full length human E-cadherin or its mutated forms was
cloned into the pMXs-IG retroviral vector, which was a kind
gift from Dr. Toshio Kitamura from the University of Tokyo.
To analyze the reprogramming efficiency by GFPþ colony
numbers, we constructed another set of vectors which do not
express the GFP protein by cutting off the GFP sequence
from the pMXs-IG backbone.
The short hairpin RNA (shRNA) targeting mouse E-cadherin
mRNA or the control shRNA was cloned into the pMKO.1 retroviral vector, which was obtained from Addgene (Addgene, Cam-
bridge, MA, http://www.addgene.org). The sequences for the
shRNA targeting mouse E-cadherin and for the control shRNA
are shown in Supporting Information Fig. S5.
Retrovirus Production and Generation of iPS Cells
Retrovirus were produced by transfection of plat-E cells with pMXs
retroviral vectors containing coding sequences of mouse Oct4,
Sox2, Klf4, and c-Myc (which were obtained from Addgene), using
FugeneHD transfaction reagent (Roche). Virus containing supernatants were added into plates of MEF cell cultures and spinned at
2,000 rpm for 90 minutes to ensure their transduction. Polybrene
was used at a final concentration of 4 lg/ml to facilitate the transduction. Medium was changed immediately after virus transduction
and this day is termed as ‘‘Day 0.’’ Two days later, medium was
changed into mES medium (DMEM supplemented with 15% FBS,
L-glutamine, nonessential amino acids, b-mercaptoethanol, and
1,000 U/ml LIF). On day 4 of post-virus transduction, transduced
MEF cultures were digested into single cell cultures and reseeded in
6-well plates at a density of 20,000 cells per well on irradiated MEF
feeders for further analysis of reprogramming efficiency.
For quantification of alkaline phosphatase-positive (APþ colonies, four factors transduced MEF cell cultures on day 10 were fixed
and stained for alkaline phosphatase activity using an alkaline phosphatase detection kit (Sigma, catalog No. 85L3R). The plates were
then photographed and the numbers of APþ colonies were counted
independently according to the photos, and at least three independent experiments completed by different lab workers in a blind manner were performed to ensure the accuracy of the final results. For
quantification of reprogramming efficiency using Oct4::GFP reporter, four factors transuded MEF cell cultures on day 16 were
scanned under a fluorescence microscope and scored for GFPþ colonies. When three factors were used, the quantification for APþ and
GFPþ were carried out on day 16 and day 20, respectively.
To generate fully reprogrammed iPS cells, ES-like colonies
were picked from transduced MEF cell cultures on day 16,
digested into single cell suspensions, and plated in 96-well plates
with mES medium. Oct4::GFP positive colonies with homogenous morphologies were further expended for characterization.
Embryoid Body Formation of iPS Cells
The iPS cells and mouse ES cells were grown to approximately
80% confluence and dissociated into single cells in the differentiation medium (DMEM supplemented with 1000U/ml leukemia inhibitory factor (LIF) and 15% FBS, L-glutamine, nonessential amino acids, and b-mercaptoethanol) and seeded at a
density of 1 105 cells per milliliter into uncoated Petri dishes.
The medium was changed every 2 days. Embryoid bodies were
collected on day 6 for real-time PCR analysis.
FACS Analysis
MEF cells were retroviral transduced with wild-type E-cadherin
or its mutants described above and incubated in DMEM with
15% FBS for 4 days. For surface staining, cells were trypsinized, washed once in PBS, and resuspended in 500 ll of 0.2%
BSA in PBS (FACS buffer) containing a rat anti-mouse E-cadherin antibody (DECMA-1; 1:500 dilution; Abcam, Cambridge,
U.K., http://www.abcam.com). The cell suspensions were incubated on ice for 30 minutes and then washed three times in
PBS, and the cells were resuspended in 500 l of FACS buffer
containing Cy3-conjugated anti-rat IgG antibody (1:200 dilution), and incubated on ice for 30 minutes. The cells were
washed three times in PBS and then fixed in 1% w/v paraformaldehyde in PBS. Cell fluorescence was analyzed using a Becton Dickinson FACScalibur cell sorter (BD Biosciences, San
Diego, http://www.bdbiosciences.com). Viable cells were gated
using forward and side scatter and all data represent cells from
this population.
Chen, Yuan, Wei et al.
1317
RT-PCR and Real-Time PCR
Total RNAs were extracted from cells using Trizol reagent
according to the manufacturer’s instructions (Sigma). RNA
was reverse-transcribed using random hexamers and M-MLV
Reverse Transcriptase (Promega, Madison, WI, http://
www.promega.com). For semiquantitative PCR analysis, the
cDNA solution was amplified for 30 cycles at an optimal
annealing temperature. For the real-time quantitative PCR
analysis, 2 PCR Mix (Sigma) and Eva Green (Biotium)
were used, with an MX3000P Stratagene PCR machine. The
relative expression values were normalized against the inner
control. Primer sequences for this section were listed in Supporting Information Table S1.
Western Blot
Cell lysates were extracted, separated on reduced SDS-PAGE,
and transferred onto nitrocellulose membrane. Primary antibodies were as follows: mouse anti-E-cadherin, mouse anti-Ncadherin, mouse anti-b-catenin (all 1:500 dilution; BD Biosciences), mouse anti-E-cadherin, rabbit anti-Nanog (both
1:200 dilution; Santa Cruz), mouse anti-b-actin (1:1,000;
Sigma). The membrane was detected by secondary fluorescent
antibody CW800 (Rockland) using an infrared imaging system (Odyssey LI-COR).
Antibody Blocking and Peptide Inhibition
On day 4 of post-viral transduction, MEF cells were reseeded
on irradiated feeders and cultured in mES medium as described
above. The next day, rat anti-E-cadherin antibody DECMA-1
and the E-cadherin peptide inhibitor SHAVSA were added at a
final concentration as described above, respectively. For antibody blocking, heat denatured DECMA-1 was used as control.
The peptide SHAVSA and its counterpart SHGVSA were prepared as reported [23]. The antibody or peptide was reapplied
every other day with each media change. Plates were scored for
APþ colonies after 5 days of treatment.
Teratoma Production and Analysis
Approximately 1 106 iPS cells were suspended in 100 ll
mES medium and injected into NOD-SCID mice to form teratomas. Three weeks after injection, teratomas were harvested,
fixed overnight with 4% paraformaldehyde, embedded in paraffin, sectioned, HE stained, and analyzed.
Production of Chimeric Mice
Zygotes were isolated from superovulated Female ICR mice
and iPS cells (with a C57/BL6 background) were injected into
the resulting blastocysts. Chimeras were produced by implantation of injected blastocysts into pseudopregnant ICR mice.
RESULTS
Chemicals That Increase E-Cadherin Expression
Enhance the Generation of iPS Cells
We established a 96-well-plate based chemical screening system for the four-factor-induced reprogramming. MEF cells
carrying a transgenic Oct4 promoter-driven GFP were used in
the chemical screening. More than 2,000 chemicals were
screened and 10 chemicals were found to enhance the formation of GFP-positive colonies (data not shown). Interestingly,
we noticed that two of the 10 positive chemicals, Apigenin
and Luteolin, had been reported to elevate the expression of
E-cadherin in some cell lines and animal models [24, 25].
However, S-allyl-L-cysteine (SAC), another chemical that
was reported to enhance E-cadherin expression in certain cell
www.StemCells.com
lines [26], was identified negative in the screening. We then
tested these results in a more detailed manner. MEF cells
transduced with four factors were cultured under the indicated
concentration of chemicals in DMEM medium for 3 days and
the protein levels of E-cadherin were tested by Western blotting. Notably, Apigenin significantly increased E-cadherin
expression to approximately 1.4-fold relative to control under
optimal concentration of 10 lM; Luteolin also significantly
increased E-cadherin expression to approximately 1.6-fold
under optimal concentration of 7.5 lM. SAC, however,
showed no effect under the concentrations tested and was
used as a negative control here (Fig. 1A). Apigenin and Luteolin could also increase E-cadherin expression in MEF cells
in the absence of reprogramming factors after 3 days treatment with each compound, as determined by RT-PCR analysis (Supporting Information Fig. S2B). The chemicals were
then added to the mES medium after the transduced MEF
cells were reseeded upon feeder cells on day 4 to monitor
their ability to affect the reprogramming efficiency (Fig. 1B).
Remarkably, addition of Apigenin or Luteolin on their optimized concentrations caused a nearly fourfold increase in the
number of APþ colonies, whereas addition of SAC showed
no obvious effect (Fig. 1C and Supporting Information Fig.
S1A). A similar fourfold increase was also seen when number
of Oct4::GFP positive colonies were counted (Supporting Information Fig. S1C). The increased iPS cell generation correlated well with the upregulation of E-cadherin protein level
(Fig. 1C, 1D). We also found that Apigenin promotes iPS cell
formation in a dose- dependent manner (Fig. 1E and Supporting Information Fig. S1B), which correlates with its ability to
upregulate endogenous E-cadherin expression (Fig. 1F). The
similar dose- dependent increase was also obvious when number of Oct4::GFP positive colonies were used to compare the
reprogramming efficiency (Supporting Information Fig. S1D).
The result of MTT assay revealed that both Apigenin and Luteolin inhibit cell proliferation, indicating that the increased
iPS generation is not achieved by facilitating cell proliferation
(Supporting Information Fig. S2A). It has been reported that
Apigenin has direct interference with Smad2, a signal transducer of Tgf-b pathway known to regulate E-cadherin expression [27, 28]. We thus explored whether these two chemicals
increase E-cadherin expression through regulating Tgf-b pathway. We found that treatment with Apigenin (10 lM) or Luteolin (7.5 lM) directly reduced Smad2 expression (Supporting Information Fig. S2C), indicating attenuated Tgf-b
signaling in MEF cells may contribute to the upregulation of
E-cadherin expression. These observations demonstrate that
E-cadherin promoting chemicals have positive effect on the
iPS cell generation.
E-Cadherin Is Upregulated During the Early
Stage of Reprogramming
E-cadherin has been previously shown to express at a high
level in ES cells and in a variety of adult stem cells in Drosophila and mice [20, 29, 30]. To test the function of E-cadherin in iPS cells, we derived stable 4F (four factors; Oct4,
Sox2, Klf4, and c-Myc) iPS cells and 3F (three factors; Oct4,
Sox2 and Klf4) iPS cells from Oct4::GFP MEF cells using
the standard protocol for retroviral transduction. Single iPS
colonies were picked and expanded into stable cell lines
(named 4F-iPS and 3F-iPS throughout the article). Both 4FiPS and 3F-iPS cells were morphologically indistinguishable
from ES cells and expressed the Oct4::GFP at a high level
(Fig. 2A). Activation of Nanog as well as endogenous Oct4
and Sox2 was thought to be a property of fully pluripotent
iPS cells [2]. We selected three clones of 4F-iPS cells and
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E-Cadherin is Critical for the Genaration of iPS Cells
Figure 1. Chemicals that upregulate E-cadherin can enhance reprogramming efficiency.
(A): Immunoblotting of E-cadherin expression in four-factor transduced MEF cells
under small-molecule treatment, with the concentrations listed. Equal volume of dimethyl
sulfoxide (DMSO) was used as control.
Quantification by densitometric analysis is
shown. (B): Schematic representation of
induced pluripotent stem (iPS) cell generation
with chemical treatment. (C): Quantification
of the efficiency of four-factor induced iPS
cell generation with small-molecule treatment. The error bars denote the standard
error derived from quantification of three separate wells of cells. **p < .01, versus control. (D): Fold change in the E-cadherin
protein level under chemical treatment. The
error bars denote the standard error derived
from densitometric analysis of three independent experiments. *p < .05, versus control. (E): Quantification of the efficiency of
four-factor induced iPS cell generation under
Apegenin (5, 10, and 15 lM) treatment. **p
< .01, versus control. (F): Fold change in the
E-cadherin protein level under Apigenin (5,
10, and 15 lM) treatment. The error bars
denote the standard error derived from densitometric analysis of three independent experiments. *p < .05, versus control.
Abbreviations: APþ, alkaline phosphatasepositive; Ctr, control;; ES, embryonic stem;
MEF, mouse embryonic fibroblast; SAC, Sallyl-L-cysteine.
two clones of 3F-iPS cells for analysis. These iPS cells showed
activation of endogenous Oct4 and Sox2, as tested by primers
specific for endogenous transcripts (Fig. 2B), and expressed the
pluripotency marker Nanog (Fig. 2B, 2C). One of these iPS cell
lines, 4F-iPS-1, was injected to blastocyst and transplanted into
pseudopregnant ICR female mice to test its ability for chimeric
formation. As expected, live chimeras were obtained, proving
the full pluripotency of this iPS cell line (Supporting Information Fig. S3A). We found that E-cadherin protein was
expressed at a similar level in these stabilized iPS cells as in
ES cells but was absent in MEF cells (Fig. 2B, 2C). Notably,
N-cadherin, another classic cadherin whose tissue distribution
is distinct from that of E-cadherin, was expressed in MEF cells
but had been shut down in pluripotent cells (Fig. 2B, 2C), indicating that an N-cadherin to E-cadherin switch occurred during
the reprogramming process.
To further clarify the function of E-cadherin in iPS cells,
subcellular localization of E-cadherin as well as several other
cell adhesion regulators was compared by immunofluorescence
study in MEF cells and iPS cells. Consistent with their spindleshaped morphology, MEF cells did not express E-cadherin and
the tight junction protein ZO-1. Instead, these cells showed
high levels of N-cadherin which mainly accumulated in the
cytosol. The cadherin binding partner b-catenin also showed a
cytosolic distribution in MEF cells. However, iPS cells derived
from MEF cells formed compatible colonies as those of mouse
ES cells, accompanied by membrane localization of E-cadherin,
b-catenin, and ZO-1, and virtually complete loss of N-cadherin
(Fig. 2D). These alterations indicated that iPS cells have undergone a transition in which four-factor transduced MEF cells
established and maintained E-cadherin-mediated cell–cell contact, a typical feature of ES cells. This observation raised the
possibility that establishment of cell–cell contact has a correlation with reprogramming and E-cadherin may be a key component that contributes to this correlation.
The sequential activations of endogenous pluripotency
markers, such as SSEA1, Oct4, and Nanog, have been proposed
to reflect the progress of reprogramming [32, 33]. To further
investigate the nature of E-cadherin in iPS cell generation, we
then explored the dynamic changes of E-cadherin expression
during the reprogramming process and compared it with those
of SSEA1 and Nanog. Surprisingly, E-cadherin was activated
as early as 2 days post-virus infection, a time point when the
four reprogramming factors started to express, and further upregulated for several days upon viral transduction, then reached
a steady level (Fig. 2E, 2F and Supporting Information Fig.
S3B). However, as a late cornerstone in the reprogramming
process, Nanog only became detectable on day 8 post-viral
transduction (Fig. 2E, 2F). During the whole reprogramming
period, upregulation of SSEA1 has been defined as an early
event [32, 33]. Immunofluorescent analysis of E- cadherin and
SSEA1 expression in four-factor transduced MEF cells demonstrated that emergence of E-cadherin protein was clearly
detected at day 4, whereas SSEA1 expression could be detected
Chen, Yuan, Wei et al.
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Figure 2. E-cadherin is upregulated during the early stage of reprogramming. (A): Morphology of 4F-iPS cells and 3F-iPS cells on MEF feeder
cells. Scale bars represent 100 lm. (B): RT-PCR analysis of pluripotency markers, E-cadherin (E-cad) and N-cadherin (N-cad) expression in 4FiPS cells, 3F-iPS cells, and mES cells. Primer sets used for Oct4 and Sox2 were specific for the endogenous transcripts. b-actin was used as a
loading control [31]. (C): Western blot analysis of E-cadherin, N-cadherin, and Nanog expression on 4F-iPS cells, 3F-iPS cells, and mES cells.
(D): Immunofluorescence staining of MEF cells, mES cell, and 4F-iPS cells for pluripotency and cell–cell contact markers. DAPI staining indicates the nuclear. Scale bars represent 50 lm. (E): Real-time PCR analysis of Oct4, Nanog, E-cadherin, and N-cadherin on the indicated days in
four-factor transduced MEF cells during the reprogramming process. b-actin was used as an internal control. The error bars denote the standard
error derived from quantification of three independent experiments. (F): Western blot analysis of E-cadherin, N-cadherin, and Nanog expression
on four-factor transduced MEF cells during the reprogramming process. (G): Immunofluorescence staining of four-factor transduced MEF cells
for E-cadherin and N-cadherin on day 6 post-virus infection. Scale bars represent 50 lm. Abbreviations: iPS, induced pluripotent stem; MEF,
mouse embryonic fibroblast; mES, moue embryonic stem; SSEA1, stage-specific embryonic antigen-1; ZO-1, zona occludens 1.
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E-Cadherin is Critical for the Genaration of iPS Cells
Figure 3. Ectopic expression of E-cadherin enhances reprogramming efficiency. (A): Representative images of APþ colonies. MEF cells were transduced with retrovirus containing four factors (Oct3/4, Sox2, Klf4, and c-Myc) plus E-cadherin or three factors (Oct3/4, Sox2, and Klf4) plus E-cadherin
and cultured in the standard iPS induction conditions. Cultures were fixed and stained for alkaline phosphatase activity (Upper panel). Parallel-transduced
MEF cells were tested for E-cadherin overexpression by Western blot using antibody specific for E-cadherin (Lower panel). (B): Quantification of APþ
colony numbers after E-cadherin overexpression. Bars represent numbers that normalized as relative colony numbers per 20,000 starting cells. The error
bars denote the standard error derived from quantification of three independent experiments. (C): Quantification of Oct4::GFP positive (GFPþ) colony
numbers after E-cadherin overexpression. As a more stringent quantification for reprogramming efficiency, GFPþ colony numbers were counted under a
fluorescence microscope. Bars represent numbers that normalized as relative colony numbers per 20,000 starting cells. The error bars denote the standard
error derived from quantification of three independent experiments. (D): Morphology of iPS cells derived by the four factors plus E-cadherin method
(E4F-iPS cells). Phase contrast photo shows an ES-like morphology of E4F-iPS cells. Antibody staining shows that E4F-iPS cells express pluripotency
markers SSEA1 and Nanog. Represent pictures of iPS cell line E4F-iPS-1 are shown. The scale bars represent 100 lm. (E): Morphology of E4F-iPS
cells maintained in medium with and without LIF. E4F-iPS cells were passaged on MEF feeder cells and maintained in þ LIF medium or – LIF medium for 3 days. Morphology (phase) and Oct4::GFP expression (Oct4::GFP) were scanned and photographed. The scale bars represent 100 lm. (F):
Real-time PCR analysis of the markers for pluripotency and all three germ layers in embryoid bodies derived from E4F-iPS cells. Results from cell line
E4F-iPS-1 are shown. Bars represent expression levels relative to that of ES cells. b-actin was used as an internal control. (G): Teratomas derived from
E4F-iPS cells. Shown are representatives HE staining pictures for ectoderm (neural tissue), mesoderm (cartilage) and endoderm (gut). Scale Bars represent 50 lm. (H): Two-week-old chimeric mice derived from E4F-iPS cells. Abbreviations: APþ, alkaline phosphatase-positive; Bry, brachyury; ES, embryonic stem; ES-EB, embryonic stem cells derived embryoid bodies; GFP, green fluorescent protein; FGF5, fibroblast growth factor 5; 3F, three factors;
4F, four factors; SSEA1, stage-specific embryonic antigen-1.
Chen, Yuan, Wei et al.
until day 6 (Supporting Information Fig. S3C). These data indicated that activation of endogenous E-cadherin marks an early
event in the reprogramming process. Interestingly, the expression of N-cadherin downregulated at day 3 and kept a reduced
expression level afterward in the entire population, showing a
contrary manner with E-cadherin, as in many other biological
processes such as epithelial-mesenchymal transition (EMT)
(Fig. 2E, 2F). Immunofluorescent study further confirmed that
E-cadherin and N-cadherin were exclusively regulated in the
reprogrammed MEF cells. In the compatible cells that underwent morphological changes, E-cadherin was expressed and
localized to the cell membrane, indicating its functional roles
here; meanwhile, N-cadherin was downregulated in these cells,
in contrast to the surrounding cells which still showed typical
fibroblast-like morphology, resulting in an explicit boundary
between these two groups of cells (Fig. 2G). These data demonstrate that an N-cadherin to E-cadherin switch occurs during the
early stage of reprogramming.
Ectopic Expression of E-Cadherin Enhances
iPS Cell Generation
We next examined whether ectopic expression of E-cadherin is
sufficient to facilitate the reprogramming process and thus promote the iPS cell generation. Four or three transcription factors
accompanied with human E-cadherin were introduced into MEF
cells with retroviral vectors. In four-factor induced reprogramming, we typically got 100 APþ colonies per 20,000 starting
cells. The overall efficiency was calculated approximately 0.5%,
similar as previously reported by other groups [34]. When human
E-cadherin was introduced together, the APþ colony number
boosted to 400 per 20,000 cells (approximately 2% of efficiency,
Fig. 3A). We reproduced the experiment and consistently got a
fourfold increase in the reprogramming efficiency (approximately
2% of efficiency in the presence of E-cadherin vs. 0.5% in the absence of it), through counting either the number of APþ or GFPþ
colonies (Fig. 3B, 3C). In the three-factor induced reprogramming, the typical efficiency we got was approximately 30 colonies per 20,000 starting cells (Fig. 3A). When E-cadherin was
added to the recipe, we consistently got a threefold to fourfold
increase in the reprogramming efficiency, by counting either the
number of APþ or GFPþ colonies (Fig. 3A–3C). These data suggest that overexpression of E-cadherin by viral transduction is
sufficient to enhance the iPS cell generation.
To address whether enhanced reprogramming efficiency by
E-cadherin overexpression could finally result in bona fide iPS
cells, we then generated stable iPS cell lines using the four factors plus E-cadherin method. These iPS cells were referred to
E4F-iPS cells throughout the article. E4F-iPS cells maintain an
ES-like morphology and show significant high expression of
the pluripotency markers AP, SSEA1, and Nanog (Fig. 3D).
The overexpression of E-cadherin was confirmed by genomic
PCR genotyping (Supporting Information Fig. S4). In vitro differentiation assays further confirmed the pluripotency of the
E4F-iPS cells. In the presence of LIF in ES medium, E4F-iPS
cells expressed high level of Oct4::GFP. In contrast, in the absence of LIF in the medium, E4F-iPS cells began to differentiate, losing the colony morphology as well as the Oct4::GFP
expression (Fig. 3E). When cultured in suspension, E4F-iPS
cells formed embryoid bodies just like ES cells. Pluripotency
markers Oct4 and Nanog were largely silenced in day 6 EBs of
E4F-iPS cells, whereas markers of differentiated cell lineages
were activated to a similar level as that in ES cells derived EBs
(ES-EBs) (Fig. 3F). Teratomas that contain all three germ
layers were formed when E4F-iPS cells were injected into
NOD-SCID mice, further confirming their differentiation ability (Fig. 3G). Finally, we confirmed whether E4F-iPS cells are
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1321
fully reprogrammed iPS cells by testing their ability to form
chimeric mice following blastocyst injection. Live chimeras
with high contribution from E4F-iPS cells were obtained,
proved the in vivo differentiation potential of these cells (Fig.
3H). Our results thus show that iPS cells generated with E-cadherin overexpression are indeed fully pluripotent iPS cells.
Ablation of Endogenous E-Cadherin Reduces
the Reprograming Efficiency
To address the role of E-cadherin in iPS cell generation, we performed comparison experiments using the standard four-factor
induction protocol. Four factors were cointroduced into
Oct4::GFP MEF cells together with either E-cadherin shRNA
or control shRNA and cultured under standard conditions.
shRNA hairpins were tested to functionally downregulate Ecadherin expression in mouse hepatocytes, which express endogenous E-cadherin (Supporting Information Fig. S5A).
Although E-cadherin expression was unaffected by the control
hairpin, E-cadherin shRNA demonstrated 80%–90% downregulation of E-cadherin expression, as tested by Western blot (Supporting Information Fig. S5A). Next we explored the functional
effect of knocking down E-cadherin on iPS cell generation. For
quantification, APþ colonies were photographed and counted
on day 10 post-viral transduction (Fig. 4A). When MEF cells
were transduced with four factors plus control shRNA, we consistently got comparable iPS cell generation efficiency as
reported, approximately 150 colonies from 20,000 starting cells
[34]. However, only approximately 30 colonies (approximately
80% decrease) were available in a paralleled experiment when
E-cadherin expression was disrupted using E-cadherin shRNA
(Fig. 4A, 4B). As a more stringent quantification standard,
Oct4::GFPþ colonies were photographed and counted on day
16 post-viral transduction (Fig. 4C). Here, we typically got 45
GFPþ colonies from 20,000 starting cells. However, 12 GFPþ
colonies from 20,000 starting cells (approximately a 75%
decrease) were generated in the E-cadherin shRNA experiment
(Fig. 4C and Supporting Information Fig. S5B). Notably, the
decreased iPS cell generation was coincidental with E-cadherin
protein level (Fig. 4A). E-cadherin has been reported to arrest
cell growth in certain cancer cell lines [35, 36]; however, no
difference in proliferation was observed between the control
and E-cadherin knockdown samples in our experiment as determined by MTT assay, indicating that the reduction in reprogramming efficiency we observed was independent of cell proliferation rates (Supporting Information Fig. S5C). To further
address that the impaired efficiency was specifically resulted
from E-cadherin knockdown, we examined whether E-cadherin
reintroduction was sufficient to rescue iPS cell generation. As
expected, reprogramming efficiency was fully restored to normal levels when human E-cadherin, which was not targeted by
mouse E-cadherin shRNA, was expressed (Fig. 4B, 4C). These
data indicate that activation of endogenous E-cadherin may be
essential for normal iPS cell generation.
Adhesive Binding Activity of E-Cadherin
Is Required for Reprograming
Our data have suggested that E-cadherin plays an important
role in the reprogramming process. To further investigate the
functional role of E-cadherin, we used the adhesion inhibitory
peptide and antibody to specifically abrogate E-cadherinmediated cell–cell contact throughout the reprogramming process. HAV containing peptide (termed SHAVSA) treatment of
mES cells has been reported to result in loss of cell–cell contact by inhibiting cadherin engagement [19, 23]. Indeed, when
cultured under 10 mM SHAVSA for 24 hours, mES cells lost
their typical colony morphology and developed a fibroblast-
1322
E-Cadherin is Critical for the Genaration of iPS Cells
Figure 4. Knockdown of endogenous E-cadherin reduces the reprograming
efficiency.
(A):
Representative images of APþ colonies. MEF cells were infected with
retrovirus containing four factors in
combination with mock, E-cadherin
(E-cad) shRNA, E-cad shRNA control, and human E-cadherin (hEcad) as indicated. After 10 days,
cells were fixed and stained for AP
activity. (B): Quantification of APþ
colonies as indicated. Expression of
E-cadherin was examined by Western blot (bottom). b-actin was used
as a loading control. The error bars
indicate the standard error derived
from quantification of three independent experiments. ***p < .001,
versus control. (C): Quantification
of Oct4::GFP positive (GFPþ) colonies on day 16 after retroviral infection. The error bars indicate the
standard error derived from quantification of three independent experiments. **p < .01, versus control.
Abbreviations: APþ, alkaline phosphatase-positive; GFP, green fluorescent protein.
like phenotype, showing the cell–cell contact had been disrupted. In contrast, SHGVSA, the malfunctioned counterpart
of SHAVSA, did not have this effect (Fig. 5A). The inhibiting
peptide was then added to iPS cell culture medium to see if it
affected the iPS cell generation process. When the concentration of SHAVSA reached 5 mM, a significant reduction in
reprogramming efficiency was observed. When the concentration of SHAVSA reached 10 mM, the reprogramming efficiency declined to approximately 60%. The control peptide
SHGVSA still exhibited no obvious effect (Fig. 5B and Supporting Information Fig. S6A).
Inhibition of E-cadherin hemophilic binding by DECMA-1
has been shown to decrease cell–cell contact in mouse ES cells
by specific blockade of the extracellular domain of E-cadherin
[29]. As reported, we observed loss of cell–cell contact in mES
cells after addition of DECMA-1 to the growth medium for 24
hours, which was similar to the effect of HAV-containing peptide SHAVSA (Fig. 5C). Therefore, we used DECMA-1 to further substantiate our conclusion. MEF cells were virally transduced with four factors and cultured under DECMA-1
containing medium. Heat-denatured DECMA-1 was used as
control. As expected, DECMA-1 decreased the number of APþ
colonies in a dosage-dependent manner. A significant decrease
was observed when 16 lg/ml of DECMA-1 was used (Fig. 5D
and Supporting Information Fig. S6B). These data further reinforce the conclusion that E-cadherin-mediated cell–cell contact
is required for efficient reprogramming.
E-cadherin itself could not only mediate cell–cell contact
but also could provide downstream signals [37–39]. In the
reprogramming process, it is rather interesting to clarify
whether this protein is only implicated in intercellular adhesion
and therefore keeps cells together, or if it is also engaged in intracellular signaling. To address this issue, we next investigated which region of E-cadherin protein was required for iPS
cell generation. ED71, the E-cadherin mutant lacking the b-catenin binding domain, EDC, the E-cadherin mutant lacking the
entire intracellular domain, and, W2A, and E-cadherin point
mutant bearing a Trp-2 to alanine mutation, which was defective in hemophilic binding, were constructed and applied in the
iPS generation recipe (Supporting Information Fig. S6C). The
properties of these mutants, including the hemophilic binding
activity, membrane localization, and stability have been wellinvestigated previously [18, 40, 41]. Expression of the E-cadherin mutants was verified by Western blotting using antibodies that recognize the intracellular domain and extracellular domain, respectively (Supporting Information Fig. S6D). As
quality control, immnofluorescence and fluorescent flow
cytometry analysis demonstrated normal cell surface expression of full length E-cadherin as well as its mutated forms
ED71, EDC, and W2A (Supporting Information Fig. S6E). We
first investigated which construct(s) could rescue the knock
down effect of E-cadherin in four-factor transduced MEF cells.
Surprisingly, ED71, the b-catenin binding domain lacking protein, could fully rescue the reprogramming efficiency compared with full-length E-cadherin, whereas EDC, the protein
lacking nearly the entire intracellular domain could also partially rescue the knockdown effect (Fig. 5E). However, the
W2A mutant exhibited no rescuing effect (Fig. 5E). We then
addressed whether these mutants would facilitate iPS cell generation when overexpressed along with the four factors. When
ED71 and EDC (the two intracelluar domain damaging proteins) were transduced together with the four reprogramming
factors, we observed a significant increase in iPS generation efficiency, the same as their full length counterpart, whereas the
W2A mutation, which shows a dominant negative effect on adhesive interactions, exhibited significant inhibition on the
reprogramming efficiency (Fig. 5F). We compared the effect
on cell proliferation of these mutants with full length E-cadherin by MTT, and no obvious difference was observed (Supporting Information Fig. S6F). Together, these data suggest
that adhesive activity of E-cadherin is critical for iPS cell
generation.
Chen, Yuan, Wei et al.
1323
Figure 5. Abrogation of cell–cell contact compromises iPS cell generation. (A): Phenotype of mES cells treated under control peptide (SHAVSA) or
with inhibiting peptide (SHGVSA) for 24 hours. Immunofluorescence analysis shows Oct4 and Nanog protein expression in peptide-treated mES cells.
Scale bars represent 100 lm. (B): Quantification of APþ colonies on four-factor transduced MEF cells treated with SHAVSA and SHGVSA. Four factors transduced MEF cells were cultured in standard ES medium or ES medium containing indicated concentrations of SHAVSA and SHGVSA peptide.
The error bars denote the standard error derived from quantification of three independent experiments. *p < .05, **p < .01, versus control. (C): Representative pictures of mES cells after treatment with the antibody DECMA-1 or the denatured DECMA-1 for 24 hours. The concentration of DECMA-1
used here is 16 lg/ml. (D): Quantification of APþ colonies on four-factor transduced MEF cells treated with DECMA-1 (4, 8, and 16 lg/ml). Heat denatured DECMA-1 (16 lg/ml) was used as control. The error bars denote the standard error derived from quantification of three independent experiments.
**p < .01, versus control. (E): Quantification of the efficiency of four-factor induced iPS cell generation after E-cadherin knockdown and rescue. MEF
cells were infected by four factors in combination with mock, E-cadherin shRNA (E-cad shRNA) and full-length (FL) E-cadherin or mutant E-cadherin
(ED71, EDC, and W2A). The number of colonies staining positive for AP (APþ) were counted on day 10 after infection. The error bars denote the
standard error derived from quantification of three independent experiments. *p < .05, **p < .01, versus control. Expression of the proteins was examined by Western blot (bottom). (F): Quantification of the efficiency of four-factor induced iPS cell generation with overexpression of FL E-cadherin or
mutant E-cadherin (ED71, EDC, and W2A). The number of APþ colonies was counted. The error bars denote the standard error derived from quantification of three independent experiments. *p < .05, **p < .01, versus control. Expression of the proteins was examined by Western blot (bottom). Abbreviations: APþ, alkaline phosphatase-positive; DAPI, 40,6-diamidino-2-phenylindole; DIC, differential interference contrast; FL, full length.
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E-Cadherin is Critical for the Genaration of iPS Cells
1324
DISCUSSION
In this study, we have identified a new route to improve the
reprogramming efficiency that involves E-cadherin-mediated
cell–cell contact. We confirm that current reprogramming
method can be optimized either by application of chemicals that
upregulate E-cadherin expression or by directly overexpression
of E-cadherin. These observations may lead to the application of
new chemicals or new factors that are involved in cell–cell contact to the current reprogramming protocol. Notably, E-cadherin
improves the reprogramming efficiency at comparable extents
between four factors and three factors induced reprogramming,
indicating its c-Myc independent mechanisms, which is similar
to many previously reported chemicals [7, 8, 10]. We noticed
that Apigenin and Luteolin have also been shown to possess
antioxidant properties [42], which may contribute to improve the
reprogramming efficiency here, as hypoxia has been reported to
enhance the generation of both mouse and human iPS cells [43].
It has been reported that the major function of E-cadherin in ES
cells is to maintain the colony morphology [21, 29]. However, our
observations reveal that E-cadherin is directly involved in the restoration of pluripotency. Interestingly, the reprogramming efficiency
is enhanced by not only the full length E-cadherin protein but also
its C-terminal truncated forms, demonstrating a new concept that
certain domains of a protein or factor is enough to facilitate reprogramming. It seems reasonable to assume that transformed MEF
cells require E-cadherin-mediated cell–cell contact to sense adhesion dependent signals from neighboring cells, for sustaining
reprogramming process [44, 45]. Candidates of the downstream
signals initiated by E-cadherin upregulation include notch signals
and gap junction dependent pathways [46–48]. Further clarifying
the responsible signals may contribute to more efficient reprogramming. Maintaining cell–cell contact is also expected to enable cells
more resistant to external and internal perturbations [49]. This aspect may be particularly important for iPS cell generation because
the reprogramming process is so complicated that any obstacle
encountered by the intermediate cells will drive them away from
the reprogramming process. The hypothesis is also supported by
the recent derivation of FAB stem cells, which are believed to be
trapped in a state of partial pluripotency because lacking of appropriate E-cadherin expression [21]. Following reprogramming, MEF
cells are believed to quickly get into the process of mesenthymal to
epithelia transition (MET) upon the expression of reprogramming
factors [50] and upregulation of E-cadherin is one of the important
features of MET [51]. Insteadly, the early upregulation of E-cadherin during reprogramming may be a direct consequence of the
expression of reprogramming factors because previously we have
found that the promoter region of E-cadherin contains Oct3/4 binding sites ([22] and unpublished data). It is reported that enhanced
MET may accelerate the reprogramming process [50]. Thus, upregulation of E-cadherin may also serve to enhance the MET transition, resulting in improved reprogramming efficiency. Our findings
therefore extended the role of E-cadherin as well as MET transition
during fibroblast to iPS cell reprogramming.
The reprogramming process is believed to be a stochastic
process amenable to acceleration through two distinct mecha-
REFERENCES
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contact may be an intrinsic property that defines the basis of
stem-cell specific identity. It is of great interest to identify the
downstream signals that contribute to this property and potentially influence the reprogramming efficiency. It is also an
opened question whether other factors and signals that function
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CONCLUSION
We have demonstrated that E-cadherin-mediated cell-cell contact is critical for the generation of induced pluripotent stem
cells. Knockdown of E-cadherin results in reduced reprogramming efficiency due to the loss of cell-cell contact. Further
more, upregualtion of E-cadherin by chemicals or by direct
overexpression significantly facilitates reprogramming. Our
results provide a novel function of E-cadherin and give new
insights into the mechanisms of reprogramming.
ACKNOWLEDGMENTS
We thank Dr. Duanqing Pei for continued help and instructions on
the iPS generation technology. We thank Dr. Junlin Guan, Dr. Jian
Zhao, and Dr. Xin Xie for helpful discussion and Jing Liao and
Lei Qian for technical assistance. We appreciate Shunmei Xin,
Shan Chen, and Xianglu Zeng for their technical assistance. We
thank all members of the lab for sharing reagents and advice. We
thank Chinese National Compound Resource Center for providing
us the chemical libraries for initial screenings. We thank FulenGen
for providing us with some of the plasmids. This research was supported by grants from the Ministry of Science and Technology
(2007CB947904, 2007CB947100, 2007CB948000, 2009CB9
41100, 2009CB940903, 2010CB944900), National Natural Science Foundation of China (30625014, 30623003, 30871285,
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08dj1400502, 09PJ1410900), Natural Science Foundation of
Shanghai (10ZR1435300), and Chinese Academy of Sciences
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