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First published online August 23, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3005 -3015
doi:10.1634/stemcells.2007-0103; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Integrins Regulate Mouse Embryonic Stem Cell Self-Renewal

Yohei Hayashia, Miho Kusuda Furueb,c, Tetsuji Okamotod, Kiyoshi Ohnumaa, Yasufumi Myoishid, Yasuaki Fukuharad, Takanori Abee, J. Denry Satof, Ryu-Ichiro Hatab,c, Makoto Asashimaa,e,g

aDepartment of Life Sciences (Biology), Graduate School of Arts and Sciences, and
bDepartment of Biochemistry and Molecular Biology, and
cOral Health Science Research Center, Kanagawa Dental College, Yokosuka, Japan;
dDepartment of Molecular Oral Medicine and Maxillofacial Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan;
eBiological Science, Graduate School of Science, University of Tokyo, Tokyo, Japan;
fMount Desert Island Biological Laboratory, Salisbury Cove, Maine, USA;
gInternational Cooperative Research Project/Japan Science and Technology Agency, Tokyo, Japan

Key Words. Embryonic stem cell • Extracellular matrix • Self-renewal • Chemically defined serum-free culture Leukemia inhibitory factor

Correspondence: Miho Kusuda Furue, D.D.S., Ph.D., Department of Biochemistry and Molecular Biology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, 238-8580 Japan. Telephone: 81-46-822-8840; Fax: 81-46-822-8839; e-mail: mihofuru{at}kdcnet.ac.jp

Received on March 14, 2007; accepted for publication on August 16, 2007.

First published online in STEM CELLS EXPRESS  August 23, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Extracellular matrix (ECM) components regulate stem-cell behavior, although the exact effects elicited in embryonic stem (ES) cells are poorly understood. We previously developed a simple, defined, serum-free culture medium that contains leukemia inhibitory factor (LIF) for propagating pluripotent mouse embryonic stem (mES) cells in the absence of feeder cells. In this study, we determined the effects of ECM components as culture substrata on mES cell self-renewal in this culture medium, comparing conventional culture conditions that contain serum and LIF with gelatin as a culture substratum. mES cells remained undifferentiated when cultured on type I and type IV collagen or poly-D-lysine. However, they differentiated when cultured on laminin or fibronectin as indicated by altered morphologies, the activity of alkaline phosphatase decreased, Fgf5 expression increased, and Nanog and stage-specific embryonic antigen 1 expression decreased. Under these conditions, the activity of signal transducer and activator of transcription (STAT)3 and Akt/protein kinase B (PKB), which maintain cell self-renewal, decreased. In contrast, the extracellular signal-regulated kinase (ERK)1/2 activity, which negatively controls cell self-renewal, increased. In the defined conditions, mES cells did not express collagen-binding integrin subunits, but they expressed laminin- and fibronectin-binding integrin subunits. The expression of some collagen-binding integrin subunits was downregulated in an LIF concentration-dependent manner. Blocking the interactions between ECM and integrins inhibited this differentiation. Conversely, the stimulation of ECM-integrin interactions by overexpressing collagen-binding integrin subunits induced differentiation of mES cells cultured on type I collagen. The results of the study indicated that inactivation of the integrin signaling is crucial in promoting mouse embryonic stem cell self-renewal.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem (ES) cells [1, 2] are pluripotent cells derived from the inner cell mass of blastocysts, which differentiate into all three germ layers. Because of this property, ES cells offer a great promise for regenerative medicine [3]. Experimental manipulation of ES cell differentiation provides a model of mammalian development that is amenable to molecular and cellular analysis [4, 5].

Experimental manipulation of ES cell self-renewal also provides a unique model of pluripotent stem cell self-renewal [6–8]. Mouse ES (mES) cell self-renewal has shown to be regulated by leukemia inhibitory factor (LIF) [9, 10]. LIF binds to a heteromeric receptor composed of gp130 and the low-affinity LIF receptor that activates STAT3, phosphatidylinositol 3-kinase (PI3K), and the mitogen-activated protein kinases (MAPK) family members ERK1 and ERK2. STAT3 plays an essential role in maintaining mES cell self-renewal [11, 12]. Recently, PI3K was reported to promote self-renewal in these cells [13]. The activation of ERK1 and ERK2 in turn negatively regulates mES cell self-renewal [14]. Thus, the self-renewal of mES cells is considered to be maintained by a balance between these signaling pathways.

Extracellular matrix (ECM) components are other important candidates for regulating ES cell self-renewal. ECM signaling is mediated largely by the integrin family of cell surface adhesion receptors, which comprise {alpha} and β subunits [15]. The interplay between ECM components and integrins offers an important function in various biological processes, including cell attachment, spreading, proliferation, survival, morphogenesis, and gene expression [16–19]. ECM components and integrins also regulate critical stages of differentiation in early embryogenesis. Fibronectin and laminin are adhesion molecules expressed at the earliest stage in mouse development and are essential for proper development [20]. Fibronectin-null embryos have mesoderm and neural tube defects [21]. Laminin-deficient embryos are unable to undergo epiblast differentiation and cavitation [22]. Members of the integrin family are expressed in spatially discrete patterns that reflect their function in early mouse embryogenesis [15]. The major receptor subunit, integrin β1, is essential for inner-cell mass development [23, 24]. Together, ECM is considered to be important in self-renewal and subsequent differentiation of ES cells, derivatives of inner cell mass [25, 26]. However, further analysis of the role of ECM components on ES cells has been hampered by undefined components from serum or feeder cells present in ES cell cultures. To understand the roles of growth factors or ECM components in mES cells, a simple, defined, serum-free culture medium designated ESF7 was recently developed that contains LIF for propagating pluripotent mES cells in the absence of feeder cells [27].

Here, to understand the role of ECM in the maintenance of mES cell self-renewal, we examined the effects of ECM components as culture substrata on the undifferentiated state of mES cell in this defined ESF7 medium. We initially hypothesized that ECM components and their integrin signals might promote mES cell self-renewal because they maintain the cell self-renewal of various somatic stem cells: epidermal stem cells [28], neural stem cells [29], and hematopoietic stem cells [30]. However, it was found that mES cells underwent differentiation on fibronectin or laminin. This differentiation was accompanied by integrin signaling activation indicated by focal adhesion kinase (FAK) phosphorylation and was blocked by neutralizing anti-integrin β1 antibody. Conversely, the stimulation of ECM-integrin interactions by overexpression of collagen-binding integrin subunits that mES cells did not express in ESF7 medium induced differentiation of mES cells. On the basis of these results, we concluded that ECM-integrin interaction negatively regulates mES self-renewal.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
The mES cell D3 line (CRL-1934; American Type Culture Collection, Manassas, VA, http://www.atcc.org) was routinely cultured in 75-cm2 plastic flasks (Corning Costar, Corning, New York, http://www.corning.com/lifesciences) coated with type I collagen (Nitta Gelatin, Inc., Osaka, Japan, http://www.nitta-gelatin.com) in a humidified atmosphere of 5% CO2 at 37°C in defined medium designated ESF7 (Cell Science & Technology Institute, Inc., Tokyo, Japan, http://www.cstimedia.com). ESF7 consisted of ESF basal medium (Cell Science & Technology Institute) supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, oleic acid conjugated with fatty acid-free bovine serum albumin (FAF-BSA; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 10 ng/ml LIF (Chemicon, Billerica, MA, http://www.chemicon.com), as described previously [27]. In the control experiments, mES cells were cultured in 25-cm2 plastic flask coated with gelatin in a conventional ES medium, complete ES medium (CEM; Specialty Media, Billerica, MA, http://www.specialtymedia.com), which consisted of Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, L-glutamine, 2-mercaptoethanol, nucleotides, nonessential amino acids, and 10 ng/ml LIF.

Cell Attachment and Proliferation
The attachment of mES cells to ECM components was measured by the procedures followed by Fassler et al. [31]. Briefly, a 96-well microplate (Corning Costar) was coated with each adhesion molecule at 4°C overnight, and then nonspecific binding was blocked with 1 mg/ml FAF-BSA (Sigma-Aldrich). ES cells were seeded at confluent density (3 x 106 cells per cm2) on adhesion molecule-coated plates in ESF7 or on gelatin-coated plates in CEM, either with or without various concentrations of anti-integrin β1 antibody (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). After 24 hours, the attached cells were fixed and stained for 30 minutes with 0.4% crystal violet (Sigma-Aldrich) in methanol. After the plate was washed and dried, a solution (1% acetic acid and 30% ethanol in water) was added to the wells to dissolve the crystal violet. The absorbance of 595 nm, which indicated the concentration of the dissolved crystal violet, was measured with a microplate reader (model 550; Bio-Rad, Hercules, CA, http://www.bio-rad.com). The following optimal concentrations of adhesion molecules were determined in the cell adhesion assays: 10 µg/cm2 type I collagen, 2 µg/cm2 type IV collagen (BD Biosciences), 2 µg/cm2 fibronectin (Sigma-Aldrich), 1 µg/cm2 laminin (Sigma-Aldrich), 10 µg/cm2 gelatin (Specialty Media), and 2 µg/cm2 poly-D-lysine (PDL) (Sigma-Aldrich) (Fig. 1A). To assess the effect of ECM components on the proliferation of undifferentiated mES cells, cells were seeded at a density of 5 x 103 cells per well in a 24-well plate coated with individual adhesion molecules in ESF7 or in a 24-well plate coated with gelatin in CEM. Cells were counted everyday with a Coulter particle counter (Beckman Coulter, Hialeah, FL, http://www.beckmancoulter.com).


Figure 1
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  		Figure 1. Attachment and proliferation of mouse embryonic stem (mES) cells cultured on various ECM components. (A): Cell attachment of mES cells to various ECM components at different concentrations. The adhesion efficiency of the cells at 24 hours in culture is shown. Each graph shows the percentage of the attached cells on each adhesion molecule in ESF7 relative to the attached cells on gelatin (4 µg/cm2) in CEM as 100% (n = 3). (B): Proliferation of mES cells on various ECM components. mES cells were seeded in a 24-well dish at 5 x 103 cells per well on each ECM component in ESF7 or on gelatin in CEM. Cells were counted every 24 hours (n = 3). Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; ECM, extracellular matrix; FN, fibronectin; GEL, gelatin; LN, laminin; PDL, poly-D-lysine.

 
Alkaline Phosphatase Activity
The alkaline phosphatase (AP) activity of mES cells cultured in the defined medium for 4 days on adhesion molecule-coated six-well plates in ESF7 or on a gelatin-coated plate in CEM was detected with a Fast Red substrate kit (Nichirei, Tokyo, http://www.nichirei.co.jp/english/index.html) as described previously [27, 32]. We counted a positive colony ratio from the observation of more than 100 colonies for each sample. We characterized a colony in which all the cells were positively stained with Fast Red as an undifferentiated colony.

Immunocytochemistry
A detailed immunocytochemical staining protocol was described previously [27]. Briefly, mES cells were fixed in 4% (wt/vol) paraformaldehyde or ice-cold acetone, permeabilized, and then reacted with primary antibodies. The primary antibodies were visualized with AlexaFluor 488-conjugated donkey anti-rabbit IgG or AlexaFluor 594-conjugated donkey anti-mouse IgG (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The primary antibodies used are listed in supplemental online Table 1. For F-actin staining, mES cells were reacted with fluorescein isothiocyanate-conjugated phalloidin (1:50; Sigma-Aldrich).

Flow Cytometry
A detailed immunostaining protocol for flow cytometry was described previously [27]. In this study, goat anti-Nanog antibody (ReproCELL, Tokyo, http://www.reprocell.com/en) was visualized with AlexaFluor 488-conjugated mouse anti-rabbit IgG (Invitrogen) or R-phycoerythrin-conjugated goat anti-rabbit IgG (Invitrogen). The primary antibodies used are listed in supplemental online Table 1.

Western Blot
To detect LIF downstream signaling, mES cells were cultured on adhesion molecule-coated six-well plates in ESF7 or a gelatin-coated plate in CEM for 2 days. To detect the activation of FAK, ES cells were inoculated at a density of 6 x 105 cells per cm2 on six-well plates coated with various adhesion molecules in the ESF basal medium and then incubated for 60 minutes. The cells were lysed in 200 µl of ice-cold lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% SDS, 1 mM Na3Vo4, 0.5% sodium deoxycholate, 5 mM EDTA, 1% Nonidet P40) and 250 µl of phosphate-buffered saline. Protein samples (25 or 50 µg) were separated in a 12.5% SDS-polyacrylamide gel and electroblotted to a polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, U.K., http://www.amersham.com). After incubation in blocking buffer for 30 minutes at room temperature, the membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies used are listed in supplemental online Table 1. The membranes were then reacted with secondary antibodies followed by horseradish peroxidase substrate according to the supplier's protocol (Pierce, Woburn, MA, http://www.piercenet.com). Protein bands on the membranes were visualized with LAS-1000 and PRO-LAS 1000 software (Fujifilm, Tokyo, http://www.fujifilm.com). The intensity of individual protein bands was quantified by densitometry using ImageJ software (NIH).

Reverse Transcription-Polymerase Chain Reaction
A detailed reverse transcription-polymerase chain reaction (RT-PCR) protocol was described previously [27]. Briefly, total RNA was extracted from ES cells cultured in an adhesion molecule-coated six-well plate in ESF7 or a gelatin-coated plate in CEM at a density of 2.5 x 103 cells per cm2 for 6 days. Quantitative PCR was performed with SYBR Green PCR Master Mix according to the supplier's directions (Qiagen, Hilden, Germany, http://www.qiagen.com) in ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The sequences of the primers for GAPDH and Fgf5 were as described previously. Relative expression of mRNAs was calculated compared with the expression in mouse whole embryos at day 10.5. PCR for integrin subunits gene expression was performed with SYBR Green PCR Master Mix according to the supplier's instructions (Qiagen). The sequences of the primers for Hnf4, Gata6, Cdx2, Hand1, Sox1, Bra, and integrin subunits are listed in supplemental online Table 2.

Transfection
The mES cells were seeded at a density of 1 x 105 cells per well in a six-well plate coated with type I collagen in ESF7. mES cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the supplier's instructions. Integrin {alpha}1 and {alpha}2 subunits expression vector consisted of whole integrin {alpha}1 (Dnaform, Yokohama, Japan, http://www.dnaform.jp/index_e.html) and {alpha}2 (Open Biosystems, Huntsville, AL, http://www.openbiosystems.com) cDNA under cytomegalovirus (CMV) promoter in pTracer-CMV2 (Invitrogen). pTracer-CMV2 was used as a mock. Transfected cells were reseeded in ESF7 with 100 µg/ml Zeocin (Invitrogen) 24 hours after transfection. These cells were used for immunofluorescence detection and RT-PCR 48 or 96 hours after transfection.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Effect of ECM on mES Cell Attachment and Proliferation
To determine the level of adhesion of mES cells to each ECM component, attachment efficiencies were calculated for cells cultured on type I collagen, type IV collagen, fibronectin, laminin, gelatin, or poly-D-lysine in ESF7. mES cells attached effectively to every ECM component, but they did not attach to tissue culture-treated plates without any ECM components in ESF7 (Fig. 1A). Then, the effect of ECM on mES cell proliferation was determined. Cells were seeded in a 24-well plate coated with individual adhesion molecules in ESF7 and in a 24-well plate coated with gelatin in CEM and were counted daily. The growth curves of mES cells cultured in ESF7 on type I collagen, type IV collagen fibronectin, laminin, gelatin, or PDL were very similar (Fig. 1B) to that on gelatin in the standard mES cell culture medium, CEM. Population doubling time was 11.1–12.1 hours. This result indicated that the individual ECM components tested had little effect on the proliferation of mES cells.

ECM Components Affect mES Cell Self-Renewal
Culturing of mES cells on the different ECM components led to variations in cell morphologies and colony shapes. Cells on type I collagen, type IV collagen, gelatin, or poly-D-lysine formed spherical colonies with poorly delineated cell-cell borders, which were typical of undifferentiated mES cells. In contrast, mES cells on fibronectin or laminin spread out like differentiated cells (Fig. 2A). For comparison, mES cells cultured on gelatin in CEM formed spherical colonies with some spreading outgrowth cells. On the basis of these results, we hypothesize that mES cells might assume various states depending on the ECM components present. Then, we measured the proportion of ES cell colonies positive for AP activity, which is widely used as an undifferentiated marker of mES cells [33]. High proportions of positive colonies were found in mES cells cultured on type I and type IV collagen, gelatin, or PDL in ESF7, but a low proportion of positive colonies was found in mES cells cultured on fibronectin or laminin in ESF7 (Fig. 2B). Immunocytochemical and flow cytometric analysis of Nanog protein expression, which is essential for the maintenance of ES cell self-renewal [34, 35], yielded results similar to those of AP staining (Fig. 2C, 2D). Immunocytochemical analysis of stage-specific embryonic antigen 1 (SSEA1) protein expression, which is used as a marker of undifferentiated mES cells [36], yielded results similar to those of AP staining and Nanog expression (Fig. 2E). No difference was found in the expression of Oct3/4 gene and protein and Sox2 gene by the various ECMs (supplemental online Fig. 1). The expression of marker genes for early differentiation was further determined by quantitative RT-PCR in cells cultured on individual ECM components. Fgf5 is expressed in the primitive ectoderm but is not expressed in the inner cell mass [37]. This gene was upregulated in mES cells cultured on fibronectin or laminin (Fig. 2F) but not in cells on collagens (types I and IV), gelatin, or PDL. Expression levels of extraembryonic marker genes (Gata6 and Hnf4 for primitive endoderm, Cdx2 and Hand1 for trophectoderm) were unchanged in these conditions. Expression levels of a neuroectoderm marker, Sox1, and a mesendoderm marker, Brachyury, were also unchanged in these conditions. Expression levels of those differentiation marker genes, except for Sox1, were slightly upregulated in mES cells cultured in CEM condition. However, we confirmed that when mES cells were cultured in CEM without LIF, the expression of these genes was much higher in mES cells than those in mES cells in CEM containing LIF, and mES cells differentiated more randomly (data not shown). These results indicated that mES cells maintained their undifferentiated state on collagens, gelatin, or PDL and that the cells began to differentiate into primitive ectoderm on laminin or fibronectin even in the presence of LIF.


Figure 2
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  		Figure 2. The effect of extracellular matrix (ECM) components on mouse embryonic stem (mES) cell undifferentiated state. mES cells were cultured in ESF7 medium on various ECM components or in CEM on gelatin. (A): Phase-contrast microphotographs of mES cells after staining of alkaline phosphatase (AP) activity. Scale bars = 50 µm. (B): Percentages of AP-positive colonies. Percentages are calculated from the observation of more than 100 colonies for each sample (n = 5). (C, D): Immunocytochemical analysis (C) and flow cytometric profile (D) of Nanog protein expression in mES cells. Scale bars = 50 µm. (E): Immunocytochemical analysis of SSEA1 protein expression in mES cells. Scale bars = 50 µm. (F): Fgf5, Hnf4, Gata6, Cdx2, Hand1, Sox1, and Bra expressions in mES cells analyzed by quantitative reverse transcription-polymerase chain reaction. Each mRNA expression level in the cells is relative to that in mouse whole embryos at 10.5 days as 1. Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; FN, fibronectin; Gel, gelatin; LN, laminin; PDL, poly-D-lysine.

 
ECM Components Modulate LIF Downstream Signaling
The above data indicated that ECM components affect mES cell undifferentiated state in a defined medium. We further hypothesized that different ECM in ES cell cultures might induce variable LIF signaling in a defined medium. The levels of LIF-induced downstream molecule activities were monitored in mES cells cultured on various ECM components in ESF7. We confirmed phosphorylated STAT3, PKB/Akt, and ERK1/2 in the cells cultured on gelatin in CEM, which is generally used to maintain undifferentiated mES cells. The ratio of phosphorylated STAT3 to total STAT3 in mES cells on fibronectin or laminin was less than half that in cells on type I collagen in ESF7 (Fig. 3A,3D). Similarly, the ratio of phosphorylated PKB/Akt to total PKB/Akt in mES cells on fibronectin or laminin was less than half that in cells on type I collagen in ESF7 (Fig. 3A,3C). The ratio of phosphorylation of ERK1/2 to total ERK1/2 in mES cells on fibronectin and laminin was more than twice than that in cells on type I collagen in ESF7 (Fig. 3A,3D). These results indicated that in the defined ESF7 medium, the effect of ECM on LIF signaling was possible to be determined and suggested that fibronectin and laminin modulated LIF downstream signaling.


Figure 3
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  		Figure 3. Activation of leukemia inhibitory factor downstream signaling in mouse embryonic stem (mES) cells cultured on various extracellular matrix components. Protein samples were lysed from mES cells cultured on each adhesion molecule in ESF7 or on gelatin in CEM for 2 days. (A): Phosphorylation level of STAT3, Akt, and ERK1/2 in mES cells was analyzed by Western blotting using antibodies to STAT3, Akt, ERK1/2, and their phosphorylated forms. (B–D): Phosphorylated STAT3 (B), phosphorylated Akt (C), or phosphorylated ERK1/2 (D) protein content quantified from the gel blot images (n = 3). Each band intensity was normalized to the total protein intensity. Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; FN, fibronectin; Gel, gelatin; LN, laminin; pAkt, phosphorylated Akt; PDL, poly-D-lysine; pERK1/2, phosphorylated ERK1/2; pSTAT3, phosphorylated STAT3.

 
Integrin Signaling Is Activated by Fibronectin and Laminin
To study the molecular mechanisms underlying ECM-associated mES cell differentiation, the expression of integrin subunits was examined. RT-PCR analysis showed that mES cells in ESF7 on all of the ECM components examined here expressed β1 and {alpha}3, 5, 6, 7, 9, and V integrin subunits (Fig. 4A). These results indicated that mES cells expressed integrin receptor complex for fibronectin ({alpha}5β1 and {alpha}Vβ1) and laminin ({alpha}3β1, {alpha}6β1, and {alpha}7β1) but not for collagens ({alpha}1β1, {alpha}2β1, {alpha}10β1, and {alpha}11β1). To determine the state of integrin activation, the phosphorylation of FAK stimulated by individual ECM components was examined by Western blotting (Fig. 4B). On fibronectin or laminin, the level of phosphorylation at Tyr-397 of FAK was more prominent than in mES cells cultured on collagens, gelatin, or PDL. The faint activation of FAK was observed in the cells on type IV collagen and PDL. It was presumed that this activation might be due to the mechanical stress generated from attaching to culture substrata [38, 39], because mES cells were observed to attach more rapidly to type IV collagen and PDL than to type I collagen and gelatin. Immunocytochemical analysis of the expression of F-actin and talin, which are associated with focal adhesions and adherens junction, showed colocalization of these proteins in cell protrusions of mES cells on fibronectin or laminin in ESF7 or on gelatin in CEM (Fig. 4C). Integrin β1 protein (Fig. 4D) and FAK protein (Fig. 4E) were also localized in the cell protrusions on fibronectin or laminin in the defined medium or on gelatin in CEM. In contrast, cell protrusions were not found in the cells on collagens, gelatin, or PDL. These results indicated that ECM-integrin signaling was activated in mES cells on fibronectin or laminin.


Figure 4
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  		Figure 4. Expression of integrin subunits in mouse embryonic stem (mES) cells. (A): The gene expression of integrin subunits in mES cells that were cultured on each adhesion molecule in ESF7 or on gelatin in CEM was analyzed by reverse transcription-polymerase chain reaction. Each mRNA expression level in the cells is relative to that in mouse whole embryos at 10.5 days as 1. (B): The expression of FAK and pFAK in mES cells was analyzed by Western blotting using monoclonal anti-FAK and monoclonal anti-pFAK antibodies. mES cells were seeded in suspension or on dishes coated with each adhesion molecule in ESF7 or on gelatin in CEM for 60 minutes and collected. (C–E): Confocal microscopy images of F-actin (green) and talin protein (red) expressions (C) and of FAK protein (green) (D) and integrin β1 protein (green) (E) in mES cells cultured on each adhesion molecule in ESF7 or on gelatin in CEM. Cells cultured for 24 hours were fixed and reacted with the antibodies. Arrowheads indicate merged focal adhesion area. Scale bar = 10 µm. Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; FAK, focal adhesion kinase; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gel, gelatin; LN, laminin; PDL, poly-D-lysine; pFAK, phosphorylated focal adhesion kinase; RT, reverse transcription.

 
The Expression of Integrin Subunits Is Upregulated During mES Cell Differentiation
We next investigated whether LIF might influence the expression of integrin subunits in response to ECM components. We had previously found that mES cells differentiate at low concentrations of LIF (0.1 ng/ml) and die without LIF [27]. Then, we compared the expression of integrin subunits mRNA in mES cells cultured in ESF7 containing 10 ng/ml LIF with that in the cells in ESF7 containing 0.1 ng/ml LIF, in which mES cells initiate cell differentiation [27]. Quantitative PCR analysis showed a higher expression of β8, {alpha}1, {alpha}8, and {alpha}11 integrin mRNA in the presence of the lower LIF concentration (Fig. 5). The expression of the other integrins did not change. These results indicated that a high concentration of LIF suppressed expressions of some collagen-receptor integrin subunits.


Figure 5
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  		Figure 5. Expression of integrin subunits in mouse embryonic stem (mES) cells cultured in ESF7 with 10 or 0.1 ng/ml LIF. The gene expression of integrin subunits in mES cells cultured on each adhesion molecule in ESF7 with 10 or 0.1 ng/ml LIF or on gelatin in CEM with or without LIF was determined by quantitative reverse transcription-polymerase chain reaction (n = 4). Each mRNA expression level in the cells was relative to that in mouse whole embryos at 10.5 days as 1. Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; conc., concentration; FN, fibronectin; Gel, gelatin; LIF, leukemia inhibitory factor; LN, laminin; PDL, poly-D-lysine.

 
Blocking the ECM-Integrin Interaction Maintains mES Cell Self-Renewal
The above results suggested that the mES cell undifferentiated state is regulated by the interaction between ECM and integrins. To further explore this possibility, a neutralizing anti-integrin β1 antibody was used to block the ECM-integrin interaction. The attachment efficiency of mES cells in ESF7 to collagens, gelatin, or PDL was not changed by the anti-integrin β1 antibody. However, the antibody markedly decreased the attachment efficiency of mES cells in ESF7 to laminin or fibronectin in a dose-dependent manner (Fig. 6A) and also increased the proportion of AP-positive cell colonies (Fig. 6B). Mouse control IgG did not affect the cell attachment or the proportion of AP-positive cell colonies (data not shown). Furthermore, to understand whether these effects may be attributed to blocking integrin signaling or to inhibiting cell attachment, fibronectin or laminin was added, together with type I collagen or PDL as culture substrata. In the presence of the anti-integrin β1 antibody, mES cells attached to these culture substrata in ESF7 (Fig. 6C). The anti-integrin β1 antibody increased the proportion of AP-positive colonies of mES cells cultured on these culture substrata (Fig. 6D). The antibody increased the cell population that was positive for Nanog protein (Fig. 6E,6F) and SSEA1 protein (Fig. 6G) and decreased the expression of Fgf5 mRNA (Fig. 6H). Therefore, the data indicate that the blocking of the ECM-integrin interaction not only inhibits the cell attachment but also supports mES cells in their undifferentiated state.


Figure 6
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  		Figure 6. Inhibition of integrin β1 in mouse embryonic stem (mES) cells. mES cells were cultured in ESF7 on each extracellular matrix (ECM) component or in CEM on gelatin with neutralizing anti-integrin β1 antibody or control mouse IgG. (A): Inhibition of cell adhesion to laminin and fibronectin by neutralizing anti-integrin β1 antibody. The adhesion efficiency of the cells cultured on each adhesion molecule in the presence of different concentrations of anti-integrin β1 antibody was plotted. Each graph shows percentage of the attached cells on each adhesion molecule relative to the attached cells on gelatin in CEM in the absence of anti-integrin β1 antibody as 100% (n = 3). (B): Percentages of alkaline phosphatase (AP)-positive colonies in the ES cells. Percentages are calculated from observation of more than 100 colonies each sample (n = 3). (C): Inhibition of cell adhesion to fibronectin and ColI, fibronectin and PDL, laminin and ColI, or laminin and PDL by neutralizing anti-integrin β1 antibody. Each graph shows the percentage of the cells attached to each ECM component mixture relative to the attached cells on fibronectin and PDL, with no anti-integrin β1 antibody as 100% (n = 3). (D): Percentages of AP-positive colonies. Percentages are calculated from the observation of more than 100 colonies of each sample (n = 3). (E, F): Immunocytochemical analysis (E) and flow cytometric profile (F) of Nanog protein expression in mES cells with 10 µg/ml neutralizing anti-integrin β1 antibody or 10 µg/ml control mouse IgG on each ECM mixture. Scale bars = 50 µm. (G): Immunocytochemical analysis of SSEA1 protein expression in mES cells cultured with 10 µg/ml neutralizing anti-integrin β1 antibody or 10 µg/ml control mouse IgG on each ECM mixture. Scale bars = 50 µm. (H): Fgf5 expression in mES cells with 10 µg/ml neutralizing anti-integrin β1 antibody or 10 µg/ml control mouse IgG on each ECM mixture analyzed by quantitative reverse transcription-polymerase chain reaction. Each relative mRNA level is relative to that of mouse whole embryos at 10.5 days as 1. Abbreviations: CEM, complete embryonic stem medium; ColI, type I collagen; ColIV, type IV collagen; FN, fibronectin; Gel, gelatin; LN, laminin; PDL, poly-D-lysine.

 
Stimulations of ECM-Integrin Interaction Inhibit mES Cell Self-Renewal
Finally, we investigated whether the stimulation of the ECM-integrin interaction inhibited the mES cell undifferentiated state. The collagen-receptor integrin {alpha}1 or {alpha}2 subunit in mES cells was overexpressed by transfection. The expression was verified by RT-PCR (Fig. 7A) and immunocytochemistry (Fig. 7B). Neither integrin {alpha}1 nor {alpha}2 subunit has been detected in the parent cells in ESF7. The proportion of AP-positive colonies decreased in the transfected cells on type I collagen but not in those on PDL, for which attachment activity is not mediated by integrin (Fig. 7C,7D). In these conditions, type I collagen substratum decreased the cell population that was positive for Nanog protein (Fig. 6E,6F) and SSEA1 protein (Fig. 6G) and increased the expression of Fgf5 mRNA (Fig. 6H) compared with PDL substrate. These results indicated that the stimulation of the ECM (type I collagen)-integrin ({alpha}1, {alpha}2) interaction inhibited the mES cell undifferentiated state.


Figure 7
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  		Figure 7. Stimulations of extracellular matrix-integrin interaction in mouse embryonic stem (mES) cells by overexpression of integrin subunits {alpha}1 and {alpha}2. mES cells were transfected with plasmid DNA (pTracer-CMV2) containing whole integrin {alpha}1 cDNA, whole integrin {alpha}2 cDNA, or empty as a mock. The transfected cells were cultured in ESF7 on ColI or PDL. (A, B): Integrin {alpha}1 and {alpha}2 expression was verified by reverse transcription-polymerase chain reaction (RT-PCR) 48 hours after transfection (A) and by immunocytochemical staining (B) 96 hours after transfection. (C, D): Phase-contrast microphotographs after staining for alkaline phosphatase (AP) activity (C) and percentages of AP-positive colonies in the transfected cells (D) 96 hours after transfection. Scale bars = 50 µm. Percentages are calculated from observation of more than 100 colonies each sample (n = 3). (E, F): Immunocytochemical analysis (E) and flow cytometric profile (F) of Nanog protein expression in the transfected cells 96 hours after transfection. Scale bars = 50 µm. (G): Immunocytochemical analysis of SSEA1 protein expression in the transfected cells cultured 96 hours after transfection. Scale bars = 50 µm. (H): Fgf5 expression in the transfected cells 96 hours after transfection was analyzed by quantitative RT-PCR. Each mRNA level is relative to that of mouse whole embryos at 10.5 days as 1. Abbreviations: ColI, type I collagen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDL, poly-D-lysine; RT, reverse transcription.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study determined the effects of ECM components on mES cell self-renewal under defined conditions. Collagens (type I and type IV), gelatin, and PDL were found to maintain the mES cell undifferentiated state. For prolonged culture, type I collagen was routinely used for 1 year (more than 50 subcultures) to maintain undifferentiated mES cells in ESF7 [27]. We have also succeeded in maintaining undifferentiated mES cells on PDL for 10 subcultures (data not shown). Ying et al. reported that mES cells could be propagated in a conventional serum-free medium on gelatin [40]. Our results and those of Ying et al. indicate that these collagens, gelatin, and PDL support sustained the culture of undifferentiated mES cells [40].

However, mES cells attached to collagens, gelatin, and PDL did not express collagen-binding integrin subunits and did not activate integrin signaling on these ECM components. Therefore, the question remains as to which molecule mediates mES cell attachment to collagens in the defined medium. We proposed that the discoidin domain receptor (DDR) [41] might serve as a collagen receptor in ESF7. Although DDR1 was expressed in mES cells, the inhibition of DDR1 by small interfering RNA (siRNA) did not change the attachment efficiency to type I collagen and the undifferentiated state of ES cells in ESF7 (unpublished data). DDR1 is not known to interact with gelatin but only with native fibrillar collagens [41]. These findings suggested that this receptor is not involved in the cell attachment. Collagens are also known to attach to cell-surface molecules through electrostatic interaction [42]. From the viewpoint that the attachment of cells to PDL is mediated by electrostatic interaction, mES cell undifferentiated state might be maintained on the ECM components that attach to cell-surface molecules through electrostatic interaction.

The results of this study demonstrated that when mES cells were cultured on fibronectin or laminin in ESF7, the AP activity and expression of Nanog and SSEA1, which are markers of undifferentiated mES cells, decreased, but the expression of Oct3/4 and Sox2 was not affected. The expression of Fgf5, which is a marker of primitive ectoderm, was upregulated in the cells. We also confirmed that the differentiation potential of embryoid bodies formed from type I collagen- and laminin-cultured mES cells in ESF7 was not compromised (supplemental online Fig. 2). All of the above phenotypes are similar to the characteristics of primitive ectoderm cells that are temporarily distinct intermediate pluripotent cell populations formed from inner cell mass and are able to differentiate into all three germ lines [43]. Although these transient cell populations were induced from mES cells [37], the reason for the mediation of this induction has not been identified. Our data suggested that ECM components and integrin signaling regulate ES cell self-renewal through this induction.

The expression of laminin subunits is upregulated during the process of primitive endoderm differentiation in vivo. These laminin subunits form a basement membrane between primitive endoderm and primitive ectoderm, which is essential for the survival and the differentiation of primitive ectoderm [44]. Fibronectin is also upregulated during the process of differentiation into primitive endoderm [45]. The deficiency of integrin β1, which is the major receptor of fibronectin and laminin, in mES cells affects morphology, adhesion, migration, and differentiation of various cell lineages but not the integration into the inner cell mass of blastocyst [31]. These reports support this study on the interplay between the ECM components (fibronectin or laminin) and mES cell differentiation [31, 44, 45]. Thus, the culture system is considered to demonstrate a novel in vitro model of early mammalian development using mES cells.

Integrin-mediated adhesion to ECM components activates MAPK signaling and sustains their activation in cooperation with cytokines [46]. It was found that integrin signaling was activated in mES cells cultured on fibronectin and laminin and that ERK1/2 was highly activated in these cells cultured on fibronectin and laminin. Activation of ERK1/2 inhibits mES cell self-renewal [14]. Therefore, the data of this study suggested that ECM-integrin signaling intensified the activation of ERK1/2 induced by LIF and consequently inhibited mES cell self-renewal. The role of downstream integrin signaling molecules in this differentiation will be an interesting avenue for future studies.

The results of this study also demonstrated that the expression of integrin subunits β8, {alpha}1, {alpha}8, and {alpha}11 was altered by reducing the LIF concentration. Sutherland et al. [47] reported that integrin heterodimers ({alpha}5β1, {alpha}6Bβ1, and {alpha}vβ3) are expressed in the mouse by the embryo throughout early development, whereas five other β1-associated {alpha} subunits ({alpha}1, {alpha}2, {alpha}3, {alpha}6A, and {alpha}7) show developmentally regulated expression. Neither {alpha}1 nor {alpha}2 was essential for the early mammal development analyzed by targeted mutants [15]. From these findings, it can be posited that LIF-induced changes in integrin expression may be accompanied by a differentiation process and not by direct regulation by LIF signaling in mES cells.

Generally, mES cells have been cultured in a medium containing serum, which is thought to enhance mES cell self-renewal [40, 48]. Serum contains ECM components, such as fibronectin and laminin [49]. The findings of this study suggest that these components negatively affect mES cell self-renewal. In fact, differentiated cells appeared spontaneously under CEM culture, and the expression of mesodermal and extraembryonic marker genes was slightly upregulated. Undifferentiated mES cells are notoriously difficult to culture without feeders in medium containing serum. This study demonstrates that a neutralizing anti-integrin β1 antibody increased AP-positive colonies under CEM culture. These results suggest that serum might impair mES cell self-renewal in the absence of feeder cells.

The experimental manipulation of human ES cells using defined culture conditions would be of enormous value to the field of regenerative medicine. To achieve this goal, simple culture substrata, such as microenvironmental molecules, are required. So far, a mixture of ECM components has been used to culture human ES cells without feeder cells [50]. Although human ES cells have different characteristics from mES cells, the findings of this study in mES cells may be helpful in developing clinical applications of pluripotent cells and understanding the mechanisms that guide cell fate decisions.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This study was supported by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.K.F., T.O., R.I.-H., and M.A.); an International Cooperative Research Project grant of the Japan Science and Technology Agency (to M.A.); a Grant from the Smoking Research Foundation (to T.O.); Grants P20-RR016463 and P30-ES03828 from the NIH (to J.D.S.); a Short-Term Fellowship from the Japan Society for the Promotion of Science (to J.D.S.); and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to Y.H.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.[CrossRef][Medline]

  2. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–7638.[Abstract/Free Full Text]

  3. Draper JS, Andrews PW. Embryonic stem cells: Advances toward potential therapeutic use. Curr Opin Obstet Gynecol 2002;14:309–315.[CrossRef][Medline]

  4. Keller G. Embryonic stem cell differentiation: Emergence of a new era in biology and medicine. Genes Dev 2005;19:1129–1155.[Abstract/Free Full Text]

  5. Pera MF, Tronson AO. Human embryonic stem cells: Prospects for development. Development 2004;131:5515–5525.[Abstract/Free Full Text]

  6. Gardner RL, Brook FA. Reflections on the biology of embryonic stem (ES) cells. Int J Dev Biol 1997;41:235–243.[Medline]

  7. Smith A. Embryonic Stem Cells. In: Marshak DR, Gardner RL, Gottilieb D, eds. Stem Cell Biology.Woodbury, NY: Cold Spring Harbor Laboratory Press,2001;205–230.

  8. Tanaka TS, Kunath T, Kimber WL et al. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 2002;12:1921–1928.[Abstract/Free Full Text]

  9. Smith AG, Heath JK, Donaldson DD et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688–690.[CrossRef][Medline]

  10. Williams RL, Hilton DJ, Pease S et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684–687.[CrossRef][Medline]

  11. Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.[CrossRef][Medline]

  12. Niwa H, Burdon T, Chambers I et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.[Abstract/Free Full Text]

  13. Paling NR, Wheadon H, Bone HK et al. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem 2004;279:48063–48070.[Abstract/Free Full Text]

  14. Burdon T, Stracey C, Chambers I et al. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol 1999;210:30–43.[CrossRef][Medline]

  15. Hynes RO. Integrins: Bidirectional, allosteric signaling machines. Cell 2002;110:673–687.[CrossRef][Medline]

  16. Boudreau N, Bissell MJ. Extracellular matrix signaling: Integration of form and function in normal and malignant cells. Curr Opin Cell Biol 1998;10:640–646.[CrossRef][Medline]

  17. Danen EH, Yamada KM. Fibronectin, integrins, and growth control. J Cell Physiol 2001;189:1–13.[CrossRef][Medline]

  18. Hata RI. Where am I? How a cell recognizes its positional information during morphogenesis. Cell Biol Int 1996;20:59–65.[CrossRef][Medline]

  19. Ramirez F, Rifkin DB. Cell signaling events: A view from the matrix. Matrix Biol 2003;22:101–107.[CrossRef][Medline]

  20. Cooper AR, MacQueen HA. Subunits of laminin are differentially synthesized in mouse eggs and early embryos. Dev Biol 1983;96:467–471.[CrossRef][Medline]

  21. George EL, Georges-Labouesse EN, Patel-King RS et al. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 1993;119:1079–1091.[Abstract]

  22. Li S, Harrison D, Carbonetto S et al. Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J Cell Biol 2002;157:1279–1290.[Abstract/Free Full Text]

  23. Fassler R, Meyer M. Consequences of lack of beta 1 integrin gene expression in mice. Genes Dev 1995;9:1896–1908.[Abstract/Free Full Text]

  24. Stephens LE, Sutherland AE, Klimanskaya IV et al. Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes Dev 1995;9:1883–1895.[Abstract/Free Full Text]

  25. Czyz J, Wobus A. Embryonic stem cell differentiation: The role of extracellular factors. Differentiation 2001;68:167–174.[CrossRef][Medline]

  26. Watt FM, Hogan BL. Out of Eden: Stem cells and their niches. Science 2000;287:1427–1430.[Abstract/Free Full Text]

  27. Furue M, Okamoto T, Hayashi Y et al. Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells. In Vitro Cell Dev Biol Anim 2005;41:19–28.[CrossRef][Medline]

  28. Zhu AJ, Haase I, Watt FM. Signaling via beta1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc Natl Acad Sci U S A 1999;96:6728–6733.[Abstract/Free Full Text]

  29. Campos LS, Leone DP, Relvas JB et al. Beta1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance. Development 2004;131:3433–3444.[Abstract/Free Full Text]

  30. Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991;352:438–441.[CrossRef][Medline]

  31. Fassler R, Pfaff M, Murphy J et al. Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts. J Cell Biol 1995;128:979–988.[Abstract/Free Full Text]

  32. Toumadje A, Kusumoto K, Parton A et al. Pluripotent differentiation in vitro of murine ES-D3 embryonic stem cells. In Vitro Cell Dev Biol Anim 2003;39:449–453.[CrossRef][Medline]

  33. MacGregor GR, Zambrowicz BP, Soriano P. Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 1995;121:1487–1496.[Abstract]

  34. Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.[CrossRef][Medline]

  35. Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.[CrossRef][Medline]

  36. Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen. Proc Natl Acad Sci U S A 1978;75:5565–5569.[Abstract/Free Full Text]

  37. Rathjen J, Lake JA, Bettess MD et al. Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 1999;112:601–612.[Abstract]

  38. Yano Y, Geibel J, Sumpio BE. Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain. Am J Physiol 1996;271:C635–C649.[Medline]

  39. Boutahar N, Guignandon A, Vico L et al. Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 2004;279:30588–30599.[Abstract/Free Full Text]

  40. Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.[CrossRef][Medline]

  41. Vogel W, Gish GD, Alves F et al. The discoidin domain receptor tyrosine kinases are activated by collagen. Mol Cell 1997;1:13–23.[CrossRef][Medline]

  42. Tye CE, Hunter GK, Goldberg HA. Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction. J Biol Chem 2005;280:13487–13492.[Abstract/Free Full Text]

  43. Pelton TA, Sharma S, Schultz TC et al. Transient pluripotent cell populations during primitive ectoderm formation: Correlation of in vivo and in vitro pluripotent cell development. J Cell Sci 2002;115:329–339.[Abstract/Free Full Text]

  44. Li S, Edgar D, Fassler R et al. The role of laminin in embryonic cell polarization and tissue organization. Dev Cell 2003;4:613–624.[CrossRef][Medline]

  45. Shirai T, Miyagi S, Horiuchi D et al. Identification of an enhancer that controls up-regulation of fibronectin during differentiation of embryonic stem cells into extraembryonic endoderm. J Biol Chem 2005;280:7244–7252.[Abstract/Free Full Text]

  46. Howe AK, Aplin AE, Juliano RL. Anchorage-dependent ERK signaling-mechanisms and consequences. Curr Opin Genet Dev 2002;12:30–35.[CrossRef][Medline]

  47. Sutherland AE, Calarco PG, Damsky CH. Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development 1993;119:1175–1186.[Abstract]

  48. Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium. Exp Cell Res 1999;247:241–248.[CrossRef][Medline]

  49. Barnes D, Sato G. Serum-free cell culture: A unifying approach. Cell 1980;22:649–655.[CrossRef][Medline]

  50. Hoffman LM, Carpenter MK. Characterization and culture of human embryonic stem cells. Nat Biotechnol 2005;23:699–708.[CrossRef][Medline]




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