Mol. Hum. Reprod. Advance Access originally published online on February 7, 2008
Molecular Human Reproduction 2008 14(3):169-179; doi:10.1093/molehr/gan001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GSK-3-specific inhibitor-supplemented hESC medium prevents the epithelial–mesenchymal transition process and the up-regulation of matrix metalloproteinases in hESCs cultured in feeder-free conditions
1Department of Embryology and Genetics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium 2Laboratory of Developmental and Tumour Biology, Université de Liège, Belgium
3Correspondence address. E-mail: urielle.ullmann{at}uzbrussel.be
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Feeder-free culture induces spontaneous differentiation of human embryonic stem cells (hESCs), identified as an epithelial to mesenchymal transition (EMT). The maintenance of pluripotency of hESCs in feeder-free cultures through the activation of the WNT pathway using a glycogen synthase kinase (GSK)-3-specific inhibitor (BIO) was reported. The aim of this study was to determine the effect of BIO on the EMT process. In contrast with those grown in feeder-free conditions with control medium, hESC colonies cultured with BIO-supplemented hESC medium did not show any fibroblast-like cells at the periphery. Transmission electron microscopy, relative quantitative real-time RT–PCR and immunostaining analyses showed the presence of epithelial features and a diminution of mesenchymal features in the BIO-treated hESCs such as a strong E-cadherin expression, the down-regulation of Vimentin, Snail and Slug expressions and a cytoplasmic β-catenin expression. An up-regulation of matrix metalloproteinases (MMP) MMP-2, MMP-9, MT-1MMP (membrane-type 1 MMP) and EMMPRIN (extracellular MMP inducer) expression was also found associated with the EMT occurring in feeder-free hESCs cultures using mouse embryonic fibroblasts conditioned medium (MEF CM). The presence of BIO clearly down-regulated the expression of these MMPs. This study showed that BIO, a GSK-3-specific inhibitor, prevents the EMT process which is associated with the feeder-free hESC culture. Nevertheless, BIO was not sufficient to expand hESCs in a long-term culture system.
Key words: epithelial–mesenchymal transition/GSK-3 inhibitor/human embryonic stem cells/feeder-free culture/matrix metalloproteinases
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Culturing human embryonic stem cells (hESCs) in feeder-free conditions on matrigelTM using conditioned medium (CM) obtained from mouse embryonic fibroblast (MEF) is known to induce a spontaneous differentiation process leading to the appearance of fibroblast-like cells at the periphery of hESC colonies (Xu et al., 2001; Rosler et al., 2004; Klimanskaya et al., 2005; Stojkovic et al., 2005; Ludwig et al., 2006). Recently, we have reported that this differentiation process recapitulates major steps of epithelial to mesenchymal transition (EMT) (Ullmann et al., 2007).
EMT is characterized by the loss of epithelial and the gain of mesenchymal features. It involves alterations in morphology, cellular architecture, adhesion and often coincides with enhanced migration properties (Guarino, 1995; Lee et al., 2006). EMT has been observed in physiological situations such as wound healing or at different stages of human embryogenesis (Nieto et al., 1994; Ciruna and Rossant, 2001; Hay, 2005). EMT also occurs in cancer cells where it is thought to be implicated in the progression of primary tumours towards metastasis (Barrallo-Gimeno and Nieto, 2005; Lee et al., 2006). The EMT process was recently described as occurring during induced differentiation of embryonal carcinoma cells (de Boer et al., 2007). It has been shown that such a spontaneous differentiation of mouse and rhesus monkey ES cells also involves EMT and it has been suggested that ESCs may be a suitable in vitro model system for the study of EMT mechanisms (Behr et al., 2005; Spencer et al., 2007).
Among the loss of epithelial features of the EMT, one observes the reorganization of E-cadherin/catenins-based cell–cell adhesion complexes which frequently involves a decrease of E-cadherin expression. Accordingly, an increased production of transcription factors such as Snail1 (Snail), Snail2 (Slug), Twist, EF1/ZEB1, SIP1/ZEB2 and/or E47 which all down-regulate E-cadherin transcription has also been largely involved in EMT processes (Hajra et al., 2002; Kim et al., 2002; Lee et al., 2006). The accumulation of cytoplasmic β-catenin, and its subsequent nuclear translocation, is also a key event of EMT. Indeed, once delocalized from the membranous complex it makes with E-cadherin and once translocated in the nucleus, β-catenin associates with members of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors family and activates specific target genes including mesenchymal markers of EMT such as Fibronectin or Vimentin (Gilles et al., 1999; Lee et al., 2006).
The level of cytoplasmic β-catenin is also regulated by the WNT pathway which activates glycogen synthase kinase 3 (GSK-3). GSK-3 is indeed a multi-tasking kinase phosphorylating specific proteins which are then degraded via their subsequent ubiquitination. After the binding of WNT protein to its receptor, the classical downstream signalling leads to the inactivation of the protein kinase GSK-3. This results in the cytoplasmic accumulation of specific proteins such as β-catenin. Following a cytoplasmic accumulation, β-catenin is transported to the nucleus where it activates the WNT target genes.
Besides this loss of typical epithelial features and the gain of typical mesenchymal markers, the overexpression of matrix metalloproteinases (MMPs), a group of extracellular matrix-degrading proteases has also been involved in the expression of a migratory phenotype by epithelial cells both in physiological and pathological EMT processes (Gilles et al., 1997; Nawrocki-Raby et al., 2003; Polette et al., 2004; Lee et al., 2006). Most MMPs are produced as biologically inactive proenzymes. The cleavage of the pro-domain is required for the generation of biologically active enzymes (Vu and Werb, 2000; Chakraborti et al., 2003). Among the 24 identified human MMPs, MMP-2 and -9 (gelatinase A and B, respectively) and MT-1MMP (membrane type 1 MMP) have been more particularly shown to play a key role in cell migration and cell invasion during EMT process (Himelstein et al., 1994; Freije et al., 2003; Fridman et al., 2003; Sato et al., 2005). MMP-2 and MMP-9 are secreted MMPs while MT-1MMP is a member of the membrane-associated MMPs and is also known as the cellular surface activator of MMP-2 (Itoh et al., 2001; Itoh and Seiki, 2006). The MMPs expression is also under control of the extracellular matrix metalloproteinase inducer (EMMPRIN; also named Basigin or CD147) (Sato et al., 1994; Biswas et al., 1995; Zucker et al., 2001).
For therapeutic perspectives, it has become a great challenge to find means to maintain pluripotency and avoid differentiation of hESC in feeder-free conditions. Recently, it has been reported that hESC medium supplemented with BIO, a GSK-3 specific inhibitor, can maintain the undifferentiated phenotype and the expression of pluripotent transcription factors in hESCs through the activation of the WNT pathway (Sato et al., 2004). Taken together with our previous observations, these data prompted us to examine the influence of BIO on the expression of EMT markers MMPs in hESCs. We have evaluated this effect through morphological studies, and through the expression of EMT markers and of MMPs by relative quantitative real-time RT–PCR, immunostaining experiments and ELISA analyses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Cultures of hESCs
Three hESC lines (VUB03_DM1 carrying the mutation for myotonic dystrophy type 1, VUB04_CF carrying a mutation in the CFTR gene and VUB07 not carrying any known genetic disorder) were at passage P75, P65 and P77, respectively, when used for our experiments. The three hESC lines were routinely maintained on MEF feeder layers with hESC medium [KnockOut Dulbecco's modified Eagle's medium (KO-DMEM) supplemented with 20% KnockOut Serum Replacement, 1 mM glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 4 ng/ml human recombinant basic fibroblast growth factor (bFGF)], as described before (Mateizel et al., 2006).
For the experiments, clumps of hESC colonies (VUB03_DM1, VUB04_CF and VUB07) grown on MEF feeder layers were obtained after cutting of colonies and careful collection with a Pasteur pipette to avoid contamination with MEF, and then equally divided between hESCs feeder-free cultures on matrigelTM using simple hESC medium, hESC medium conditioned on MEF (MEF CM), BIO-supplemented MEF CM or BIO-supplemented hESC medium (BIO medium). The MEF CM was prepared as described before (Ullmann et al., 2007). The BIO medium was composed of hESC medium supplemented with 2.5 µM BIO (GSK-3 inhibitor IX, (2'Z,3'E)-6-Bromoindirubin-3'-oxime, Calbiochem, Darmstadt, Germany, http://www.calbiochem.com). This concentration was chosen after comparison of concentrations ranging from 1 to 5 µM. The osmolarity of both the BIO and the hESC medium was around 635 mosm/ml. The hESCs were plated on a six-well plates (Nunc) coated with growth-factor-reduced-matrigelTM matrix (BD, Bedford, MA, USA; http://www.bdbiosciences.com, 1:30 dilution in KO-DMEM) and incubated at 37°C in 10% CO2 air in a fully humidified atmosphere. Media were replaced each day. Passaging of hESCs was carried out every 6 days after incubation with collagenase IV solution (1 mg/ml in KO-DMEM, Invitrogen) for 5–10 min.
Short culture periods were necessary to avoid confluent cell culture, which could interfere with the study of the early differentiation process. For transmission electron microscopy experiments and for immunostaining experiments, hESC colonies were plated on matrigelTM-coated four-well chamber-slides (Sonic Seal Nalgene Nunc International, Rochester, NY, USA, www.nalgenenunc.com) and allowed to expand until Day 3. For real-time RT–PCR analyses, hESCs were plated on six-well plates (Nunc) and collected at Day 3. Each experiment was repeated at least three times with the same hESC line.
Transmission electron microscopy
Sample preparation for microscopy studies was carried out as described before (Ullmann et al., 2007). The TEM analysis was carried out with at least 
200 hESC colonies plated on a four-well chamber-slide. This experiment was repeated three times.
RNA isolation and relative quantitative real-time RT–PCR
A collagenase IV solution (1 mg/ml in KO-DMEM) was applied during 5–10 min to hESCs cultured in feeder-free conditions and during 1 h to hESC cultured on feeder layers at 37°C to ascertain that all the hESCs present in each dish were collected. We collected the hESC cells plated in 2 wells of a six-well plate for each culture condition and each experiment was repeated at least three times. This method was applied for the three hESC lines used in the study. We can consider that 200 hESC colonies were plated in each well. For some experiments, the distinct cell populations of the hESC colonies within a feeder-free culture, i.e. cells from the centre (epithelial-like) or from the periphery (mesenchymal-like), were collected separately using a Pasteur pipette as described earlier (Ullmann et al., 2007).
RNA extraction was carried out with the RNeasy kit (Qiagen, Hilden, Germany) followed by treatment with the RNase-free DNase kit (Qiagen). Five micrograms of total RNA were reverse-transcribed by using the first strand cDNA synthesis kit (Amersham Biosciences, Buckinghamshire, UK, www.amershambiosciences.com) with the NotI-d(T)18 primer according to the manufacturer's instructions.
Relative quantitative real-time RT–PCR was performed on the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, USA, www.appliedbiosystems.com). The final reaction volume of 25 µl contained 12.5 µl of 2x TaqMan Universal Master Mix (Applied Biosystems), 1.25 µl of 20x Assays-on-demand Gene Expression assay mix (Applied Biosystems) and 10–100 ng cDNA in 11.25 µl nuclease-free water. The primers and the probes for the gene expression analyses of E-cadherin, Vimentin, Snail (Snail1), Slug (Snail2), POU5F_iA, MMP-2, MMP-9, MT-1MMP, EMMPRIN and GAPDH were purchased from Applied Biosystems (assays on demand gene expression products, Applera International Inc, Pleasanton, USA). The following conditions were used: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C. All experiments were run in triplicate and control samples with cDNA derived from mouse fibroblasts were taken along in the different assays to check for a possible contamination with murine mRNA in the real-time RT–PCR experiments. Relative quantification of gene expression between multiple samples was achieved by normalization against the endogenous control GAPDH using the 
Ct method of quantification. Fold changes were calculated as 2–Ct.
Immunocytochemistry analyses
In order to test for the presence of POU5F1_iA, β-catenin, N-cadherin, MMP-2, MMP-9, MT-1MMP and EMMPRIN, the hESCs cultures were fixed on matrigelTM-coated four-well chamber-slides in 4% formaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Following washing steps with PBS (3 x 5 min), incubation in 3% bovine serum albumin (Calbiochem, Darmstadt, Germany; http://www.calbiochem.com) was performed during 30 min to avoid non-specific reactions. Incubations with mouse primary antibodies were carried out at room temperature for 1 h with antibodies against POU5F1_iA (IgG2b OCT-3A (C-10) sc-5279, Santa Cruz Biotechnology Inc., Heidelberg, Germany; http://www.scbt.com, 1:50 dilution in 1.5% BSA), for β-catenin (IgG3 clone 196618, R and D systems, Minneapolis, USA, http://www.rd-systems.com, 1:100 dilution in 1.5% BSA), for N-cadherin (IgG1 clone 32, Becton Dickinson Transduction Laboratories, Lexington, KY, USA, http://www.bdbiosciences.com, 1:50 dilution in 1.5% BSA), for MMP-2 (IgG1, clone CA-4001, Lab Vision corporation, Fremont CA, USA, http://www.labvision.com, 1:50 dilution in 1.5% BSA), for MMP-9 (IgG1, clone GE-213, Lab Vision corporation, 1:50 dilution in 1.5% BSA), for MT-1MMP (IgG1, clone 5H2, R and D systems, Abingdon, UK, wwwrndsystems.com, 1:50 dilution in 1.5% BSA) and for EMMPRIN (IgG1, clone HIM6, research diagnostics Inc, Flanders NJ, USA, RDI, http://www.researchd.com, 1:100 dilution in 1.5% BSA). The primary antibodies were detected with Alexa Fluor 488-conjugated F(ab')2 fragment of goat anti-mouse IgG (H+L) (clone A11017
[GenBank]
, Invitrogen; 1:100 dilution in 1.5% BSA).
For NANOG and for Connexin 43 immunostainings, rabbit polyclonals were used: anti-NANOG (clone ab21624, Abcam, Cambridge, UK, http://www.abcam.com, 1:50 dilution in 1.5% BSA) and anti-Connexin 43 (clone C6219, Sigma, 1:100 dilution in 1.5% BSA). Detection was carried out using the secondary antibody Alexa Fluor 488-conjugated F(ab')2 fragment of goat anti-rabbit IgG (H+L) (clone A11070 [GenBank] , Invitrogen, 1:100 dilution in 1.5% BSA).
For the E-cadherin/Vimentin double immunostainings, a fixation with cold methanol (at 4C°) was applied for 1 min. After blocking with 3% BSA, incubation with a mouse primary antibody for Vimentin (IgG1, clone V9, Sigma; Saint-Louis, USA; http://sigma-aldrich.com, 1:30 dilution in 1.5% BSA), was performed at room temperature for 1 h. The secondary antibody Texas Red-conjugated F(ab')2 fragment of goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories Inc, West Grove, USA, http://www.jacksonimmuno.com, 1:100 dilution in 1.5% BSA) was applied for 1 h after washing steps. A final incubation was performed with fluorescein labelled E-cadherin antibody (mouse IgG2a, clone 36, Becton Dickinson Transduction Laboratories, 1:50 dilution in 1.5% BSA) for 1 h.
Samples in which the primary antibodies were omitted or replaced with a mouse immunoglobulin isotype used at the same dilution as the primary antibody served as negative controls: HLA-DR IgG2b antibody (clone TÜ36, BD Biosciences) for POU5F1_iA, HLA-G IgG1 antibody (MEM-G/9, Exbio, Prague, Czech Republic; http://www.exbio.cz) for Vimentin, MMP-2, MMP-9, MT1-MMP and for EMMPRIN, HLA-DR IgG3 (HL39, Exbio) for β-catenin, fluorescein labelled IgG2a antibody (clone X39, BD Biosciences) for fluorescein labelled E-cadherin and rabbit IgG (Invitrogen) for Connexin 43 and for NANOG. The images were scanned by confocal microscopy with an Argon–Krypton laser (488/568) (Fluoview IX70; Olympus, Belgium).
MMP-2 and MMP-9 ELISA
Similar amounts of cells (around 220 hESC colonies/well) were plated in a six-well plate at Day 0 and they were cultured for 4 days in feeder-free conditions using either MEF CM or BIO medium. We collected CMs from hESC cultures and we repeated this experiment at least three times for each hESC line (VUB03_DM1, VUB04_CF and VUB07). Because culture medium with serum replacement could contain some MMPs which could interfere with MMP ELISA analyses, the medium was replaced with 2 ml of serum replacement-free MEF CM or with 2 ml of serum replacement-free BIO medium, respectively, on the day before collecting the sample for ELISA analysis. The samples were conditioned during 24 h on hESCs and were collected at a different day of culture (Day 2, 3 and 4). After a 1000 rpm centrifugation, the supernatants were immediately frozen and stored at –20°C for ELISA. HESCs were detached with trypsin (0.05% in PBS) and centrifuged. A haemocytometer was used to determine the cell number after resuspending the pellet in 1 ml of medium and staining with Tryptan blue.
The determination of the concentration of secreted human MMP-2 and MMP-9 forms in the supernatants was performed by ELISA, according to the manufacturer's instructions (MMP-2 Human Biotrak ELISA system RPN2617, and MMP-9 Human Biotrak ELISA system RPN2614, Amersham Biosciences) and the plate was read at 450 nm by Easia Reader. Negative control samples were serum replacement-free MEF CM or serum replacement-free BIO medium.
Statistical analysis
Each experiment was repeated at least three times. The presented values are given as means ± SD. Differences between groups were determined by paired t-test. A P-value of less than 0.05 was considered as significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Influence of BIO medium on the morphology of hESC colonies
We have recently reported that a few days after switching hESC colonies from culture on MEF feeder layers to feeder-free conditions on matrigelTM using MEF CM, mesenchymal-like cells appear at the periphery of the colonies corresponding to an EMT process. This feeder-free culture system maintains the pluripotency of the hESCs and allows the culture for 37 passages (Ullmann et al., 2007).
Culturing hESCs in feeder-free conditions on matrigelTM with hESC medium leads to a complete differentiation of all colonies followed by cell death after few days. The fibroblast-like cells appear first at the periphery of the colonies and then the differentiation process further extends into the centre of the colonies. After 3 days, hESCs show fully flattened morphology (Fig. 1A and B). This spontaneous differentiation process is in our study demonstrated to be associated with characteristic EMT events including loss of E-cadherin expression, increased Vimentin and N-cadherin expression, nuclear staining of β-catenin (Fig. 1C–F), up-regulation of Snail and Slug expression (data not shown) and a spreading of the fibroblast-like cells.
|
We tested the addition of different BIO concentrations to the MEF CM and to the hESC medium within the feeder-free culture system using matrigelTM coating. In our hands, only a concentration of 2.5 µM BIO gave an optimal adhesion of the hESC colonies and this concentration was used through this study. A higher concentration (5 µM BIO) gave rise to hESC colonies that were too compacted and showed a low adhesion during the days following plating. HESCs treated with a lower concentration (1 µM BIO) showed a comparable morphology to that of hESCs cultured with MEF CM.
When culturing hESC colonies using MEF CM medium supplemented with 2.5 µM BIO, most of the hESC colonies (around 60% of total) showed clearly defined borders without any mesenchymal-like cells, the rest of the hESC colonies underwent an EMT process at their periphery (Fig. 2A). The EMT markers expression study demonstrated a strong E-cadherin expression and positive cytoplasmic β-catenin expression within most of the colonies and N-cadherin and Vimentin positive mesenchymal cells were found at the borders of early differentiated colonies (data not shown).
|
When culturing hESC colonies using hESC medium supplemented with 2.5 µM BIO (BIO medium), most of the hESC colonies (around 90% of total) formed small round and compacted colonies; a minority of colonies showed a less compacted morphology but still had clearly defined borders (Fig. 2B). No mesenchymal-like cells were observed (Fig. 2C). After 6–7 days, hESC colonies cultured with BIO medium maintained their undifferentiated morphology but further feeder-free culture was impossible as most of colonies could not attach and grow after few passagings.
Because, as explained above, all cells quickly underwent EMT in hESC control medium and died, we have chosen to further study the effect of BIO medium in the feeder-free culture conditions but compared with the MEF CM. These culture conditions indeed allowed the survival of the cells because the differentiation process only affects cells at the periphery of the colonies and thus represent a more valuable control for our further investigations.
We made ultra-thin sections of plastic-embedded hESC colonies to study their morphology in more detail. As previously reported, three different morphological zones were observed in hESC colonies cultured with MEF CM (Ullmann et al., 2007), a single upper layer of columnar cells facing the medium (zone 1) which covers a multilayered core of small rounded cells (zone 2) and a zone of mesenchymal-like EMT-derived cells at the periphery (zone 3), that we showed to be derived from cells of zone 1 trough EMT process. In the presence of BIO medium, most of the colonies were composed of zone 1 and zone 2 only and no zone 3 was observed. At the ultrastructural level, the columnar cells (zone 1) showed epithelial-like characteristics with a clear polarization, cell junctions at the apical side and microvilli (Fig. 2D). The small rounded cells of the central core (zone 2) showed a high nuclear-cytoplasm ratio and were closely apposed (Fig. 2E); some cells were in metaphase (Fig. 2F). Transmission electron microscopy confirmed the absence of mesenchymal-like cells at the periphery (zone 3). This suggests that the previously described EMT process, shown to be associated with these mesenchymal-like cells, does not occur when hESC medium supplemented with BIO is used.
Influence of BIO medium on the expression of EMT markers in feeder-free conditions
Relative quantitative real-time RT–PCR analyses
To better characterize the relation between the BIO medium and the EMT process, we examined the expression of several EMT markers (E-cadherin, Vimentin, Snail and Slug) and of the pluripotent stem cell marker (POU5F1-iA) in samples from hESCs (VUB03_DM1, VUB04_CF and VUB07) cultured in feeder-free conditions and comparing BIO medium with MEF CM (Fig. 3).
|
It has been shown that switching hESCs from culture on feeder layers to feeder-free conditions using MEF CM was characterized by a decrease of E-cadherin expression and an increased expression of Vimentin as well as Snail and Slug (Ullmann et al., 2007). The mRNA levels of E-cadherin in BIO-treated cells at Day 3 of culture were 4.6-fold up-regulated whereas the expression levels of Vimentin, Snail and Slug were down-regulated at 0.3-fold, 0.06-fold and 0.16-fold, respectively, when compared with hESCs cultured with MEF CM. POU5F1-iA expression was slightly decreased after switching from culture on feeder layers to feeder-free with MEF CM, whereas the mRNA levels of POU5F1-iA were slightly up-regulated (1.33-fold) in samples of BIO-treated cells (Fig. 3).
The control samples were found to be positive for Vimentin only. Although contamination with murine mRNA is very unlikely because care was taken to collect hESCs only, contamination with murine mRNA in samples would approximately be equal and would not lead to an underestimation or an overestimation of the Vimentin mRNA level in both feeder-free conditions.
Immunostainings with EMT markers antibodies
We next performed immunostaining for E-cadherin and Vimentin, chosen as specific markers for epithelial and mesenchymal cells, respectively (Lee et al., 2006). In the hESC colonies cultured with MEF CM, E-cadherin was weakly expressed with a less structured web-like structure in the membrane of hESCs found in the upper layers (zone 1) of the colonies and largely disappeared in the lower layers (zone 2) of the colonies after scanning with a confocal microscope (Fig. 4A). When hESCs (VUB03_DM1, VUB04_CF and VUB07) were cultured using BIO medium, E-cadherin was strongly expressed as a web-like structure at the membrane level of the upper layer cells and was also detected in the lower layers of the colonies (Fig. 4B). In contrast to the hESC colonies cultured with MEF CM showing Vimentin-positive mesenchymal-like cells at the periphery (zone 3) (Fig. 4C), Vimentin was barely detectable in the colonies treated with BIO medium (Fig. 4D).
|
Further analysis of the expression of the EMT markers involved immunostaining for Connexin 43 and β-catenin. Connexin 43 is a protein forming functional gap junctions and β-catenin is a cadherin-associated protein involved in the regulation of the membrane structure and epithelial cell adhesion (Wong et al., 2004). Nuclear accumulation of β-catenin is another typical change during EMT (Muller et al., 2002; Maeda et al., 2005; Lee et al., 2006). Immunostaining for Connexin 43 of hESCs cultured with MEF CM showed a strong expression at the membrane level for the centre of the colonies whereas a weaker cytoplasmic staining was found in the mesenchymal-like cells at the periphery (Fig. 4E). BIO treated hESC colonies showed an intensive staining at the membrane level of the upper layer cells, whereas the cells in the centre of the colonies displayed a weaker Connexin 43 expression at the membrane level (Fig. 4F). Immunostaining for β-catenin of hESCs cultured with MEF CM showed a strong expression at the membrane level in the centre of the colonies while a nuclear staining was found in the mesenchymal-like cells at the periphery (Fig. 4G). In the presence of BIO, a strong β-catenin positive staining was found at the membrane level of the upper layer cells whereas a positive cytoplasmic and nuclear expression was detected within the central layers (Fig. 4H).
In the hESC cultured with MEF CM, immunostaining for pluripotency stem cell markers POU5F1_iA (formerly called OCT-3A) (Cauffman et al., 2006) and NANOG (Hyslop et al., 2005) showed a positive staining in small, rounded nuclei of the central parts and a decreased intensity in the larger, irregular nuclei of the mesenchymal-like cells at the periphery (Fig. 4I and K). BIO treated hESC colonies showed a positive expression of POU5F1_iA and of NANOG in all the cells, all showing small, rounded nuclei (Fig. 4J and L).
Influence of BIO medium on the expression of MMPs
Relative quantitative real-time RT–PCR analyses
Several MMPs have been shown to play a key role in tumour-associated EMT (Lee et al., 2006). This prompted us to analyse the expression level of these particular MMPs in different hESCs culture conditions in order to investigate the relation between MMPs expression and the EMT process occurring during hESCs feeder-free culture and leading to the appearance of fibroblast-like cells at the periphery of the colonies.
We first examined the expression level of MMP-2, MMP-9, MT-1MMP and EMMPRIN genes by relative quantitative real-time RT–PCR in hESCs (VUB03_DM1, VUB04_CF and VUB07) cultured in feeder-free conditions using MEF CM compared with hESCs cultured on feeder layers (Fig. 5A). The mRNA levels of MMP-2, MMP-9, MT-1MMP and EMMPRIN were up-regulated at 3.8-fold, 2.7-fold, 7.5-fold and 1.4-fold, respectively. Within hESC colonies feeder-free cultured with MEF CM, analysis of samples enriched in mesenchymal-like cells present at the periphery revealed 2.6-fold, 2.1-fold and 3.6-fold up-regulations of mRNA levels of MMP-2, MMP-9 and MT-1MMP respectively, when compared with the samples enriched with the central parts of these feeder-free cultured hESC colonies (Fig. 5B). The EMMPRIN mRNA levels were similar in every part of the hESC colony.
|
We subsequently carried out relative quantitative real-time RT–PCR analyses for samples from BIO-treated hESCs to study the influence of BIO medium on the MMPs expression levels (Fig. 5C). The mRNA levels of MMP-2, MMP-9 and MT-1 MMP were down-regulated at 0.7-fold, 0.08-fold and 0.2-fold, respectively, whereas the mRNA levels of EMMPRIN were approximately equal when compared with samples from hESCs feeder-free cultured with MEF CM (Fig. 5C).
The control samples were found to be positive for EMMPRIN only. Although contamination with murine mRNA is very unlikely because care was taken to collect hESCs only, contamination with murine mRNA in samples would approximately be equal and would not lead to an underestimation or an overestimation of the EMMPRIN mRNA level in both feeder-free conditions.
Immunostainings with MMPs antibodies
In order to study the MMP expression at the protein level, we performed immunostainings with antibodies against MMP-2, MMP-9, MT1-MMP and EMMPRIN (Fig. 6). In hESC colonies feeder-free cultured with MEF CM, a positive cytoplasmic expression of MMP-2 and MMP-9 was found both in the central parts and in the mesenchymal-like cells at the periphery (Fig. 6A and C). In contrast, a weaker expression pattern for MMP-2 and MMP-9 was found in BIO-treated hESC colonies (Fig. 6B and D). Whereas hESCs cultured in MEF CM displayed a positive expression of MT1-MMP at the membrane level of the central parts of hESC colonies and a nuclear localization in few mesenchymal-like cells present at the periphery (Fig. 6E), a very weak expression of MT1-MMP was found in hESC colonies feeder-free cultured with BIO medium (Fig. 6F). A stronger positive expression of EMMPRIN was found at the membrane level of hESC colonies cultured in MEF CM compared with the hESC colonies cultured in BIO medium (Fig. 6G and H).
|
MMP-2 and MMP-9 ELISA analyses
In order to evaluate the impact of BIO medium on the secretion of MMP-2 and MMP–9 forms by hESCs (VUB03_DM1, VUB04_CF and VUB07), specific ELISA tests were carried out. The values of secreted MMP-2 (non-active and active forms) in the medium conditioned on hESCs cultured in MEF CM were detected around 57.2 ng/ml at Day 2 and the time course experiments revealed increasing concentrations (64.7 ng/ml and 106.2 ng/ml at Day 3 and 4, respectively) (Fig. 7A). For the hESCs cultured in BIO medium, significantly lower MMP-2 concentrations were detected (7.7, 23.1 and 29.4 ng/ml at Day 2, 3 and 4, respectively) (Fig. 7A). The values of secreted MMP-9 forms in the medium conditioned on hESCs cultured in MEF CM were positive but extremely low (around 0.6 ng/ml at Day 2) and the time course experiments revealed a stabilization of the MMP-9 values (around 0.3 ng/ml) (Fig. 7B). For the hESCs cultured in BIO medium, the MMP-9 concentration was below the sensitivity of the MMP-9 ELISA kit (Fig. 7B). The control samples were negative for MMP-2 and MMP-9 after ELISA analyses.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
It is known that culturing hESCs in feeder-free conditions in the absence of fibroblast CM induces the complete differentiation of all colonies into fibroblast-like cells, ultimately leading to cell death (Xu et al., 2001). The results of this study suggest that the addition of BIO prevents this early differentiation process including EMT characteristics. Furthermore, the addition of BIO to hESC cultures with MEF CM also prevents the EMT process which, in this case, only extends to the periphery of the colonies. The use of BIO medium in feeder-free culture conditions allowed hESC proliferation with the maintenance of the undifferentiated morphology.
Concerning the EMT markers, the RT–PCR results and the immunostainings of the BIO-treated colonies revealed a higher expression of the epithelial marker E-cadherin, and a lower expression of the mesenchymal marker Vimentin, when compared with colonies feeder-free cultured with MEF CM. Moreover, the expression levels of Snail and Slug, which are repressors of E-cadherin, were found to be drastically decreased within the BIO-treated hESC colonies, suggesting the absence of an EMT process. The Connexin 43 immunostainings suggested functional gap junctions and the β-catenin expression pattern within the BIO-treated hESC colonies showed a positive expression at the membrane level inside the upper cell layer whereas a cytoplasmic and nuclear expression was found within the central multilayered core. A β-catenin staining limited to the nucleus was detected within mesenchymal-like cells present at the periphery of hESC colonies feeder-free cultured with MEF CM, suggesting a transcriptional role of β-catenin within hESCs undergoing EMT (Ullmann et al., 2007).
Though our data are in agreement with those of Sato et al. (2004) showing that BIO prevented the differentiation of hESC cells, they somehow contrast in different aspects. Indeed, our observations concerning the β-catenin staining are in disagreement with their results showing a positive β-catenin staining limited to the nucleus inside the BIO-treated colonies (Sato et al., 2004). The reason for this discrepancy is unknown; but the role of the β-catenin pathway in hESC differentiation appears up to now controversial. If GSK-3 has clearly been shown to regulate the level of cytoplasmic β-catenin, it is still unclear how this affects it subcellular localization and thus its transcriptional activity. In this context, it is very likely that the source of hESC, the culture conditions and the confluence are factors which very likely affect BIO-dependent β-catenin regulation. Indeed, also contrasting with the results from Sato et al. (2004), Dravid et al. (2005) described that the WNT/β-catenin transcriptional activity is low in pluripotent hESCs and up-regulated in differentiated cells.
Several extrinsic signals such as bFGF, bone morphogenesis protein (BMP) and WNT support the self-renewal and pluripotency of hESCs cells through regulating the pluripotent genes (Xu et al., 2005a,b) but WNT activation alone does not seem sufficient to maintain the self-renewal and the pluripotency of the hESCs (Dravid et al., 2005). Indeed, although the BIO-treated hESC colonies displayed an epithelium-like morphology with positive pluripotent stem cell marker expression (POU5F_iA and NANOG) as described by Sato et al. (2004), we did not succeed in prolonging the hESC culture with subsequent passages. These last results are very similar to the results obtained by Dravid et al. (2005) and are totally contradictory to the main conclusion of Sato et al. (2004).
Although controversy exists, WNT/β-catenin signalling has been demonstrated to maintain pluripotency in stem cells under certain culture conditions and is critical for expansion of progenitors (Willert et al., 2003; Lowry et al., 2005; Miyabayashi et al., 2007). Additional information about long-term expansion of hESCs via activation of WNT pathway is provided by a recent paper elucidating the role of WNT/β-catenin in mouse ESC pluripotency (Miyabayashi et al., 2007). The main effect of the activation of the WNT pathway, using WNT proteins (Dravid et al., 2005), inhibitor of GSK-3 (BIO) (Tseng et al., 2006) or molecule increasing β-catenin/CBP transcription (IQ-1) is the stimulation of hESCs proliferation. While the maintenance of hESCs undifferentiated state (Sato et al., 2004; Miyabayashi et al., 2007) or the induction of hESCs differentiation (Dravid et al., 2005; Tseng et al., 2006) is probably determined by other factors. Taken together, these data clearly indicate a role of the β-catenin pathway in hESC fate but this role is obviously complex and requires further extensive characterization.
One also has to keep in mind that, as a GSK-3-specific inhibitor, BIO is indeed likely to regulate the steady state level of many other known or not yet unidentified proteins. Among the known proteins which are regulated by GSK-3 and which are implicated in EMT pathway is Snail. Snail, a transcription factor repressing E-cadherin, was indeed recently found to be regulated by GSK-3 (Zhou et al., 2004; Bachelder et al., 2005; Peinado et al., 2005). The involvement of GSK-3 in the regulation of Snail is particularly interesting because GSK-3 is known to be involved in the regulation of protein kinase AKT, FGF, WNT and hedgehog pathways (Doble and Woodgett, 2003). These pathways are known to control cell fate during development and tumorigenesis (Zhou and Hung, 2005; Katoh and Katoh, 2006; Lee et al., 2006; Thiery and Sleeman, 2006). Nevertheless, Snail regulation by GSK-3 has also been shown to be dual. Indeed, several studies showed that GSK-3 inhibits Snail activity through the control of protein stability and cellular localization (Zhou et al., 2004; Peinado et al., 2005). On the other hand, GSK-3 inhibition was also shown to enhance Snail expression through the activation of NF-kB, a well-known transcription factor also involved in EMT process (Bachelder et al., 2005). Such a dual regulation of Snail by GSK-3 further establishes the complexity of the regulation of hESC differentiation/proliferation by the GSK-3 network. This also emphasizes that common signalling pathways are involved both in EMT process and in the maintenance of hESCs pluripotency and that triggering a hESC towards EMT differentiation or pluripotency may require a fine control of regulatory at the level of GSK-3.
More than studying the classical markers of EMT, we have also originally investigated in our hESC systems the expression of several MMPs, known to contribute to tumour-associated EMT. Our study thus demonstrates a positive expression of MMPs and EMMPRIN within undifferentiated hESCs and hESCs undergoing EMT. When the hESC colonies were cultured with BIO medium, decreased expressions of MMPs were found suggesting not only that BIO prevents the up-regulation associated with the EMT process but also a key role of MMPs in this EMT process. Accordingly, MMPs and their regulator EMMPRIN were described to play a key role in pre-implantation embryogenesis and in peri-implantation development (Igakura et al., 1998; Zhou et al., 2000; Wang et al., 2003) and high MMPs gene expression has been previously reported in human adult mesenchymal stem cells where they are involved in differentiation, proliferation and migration (Mannello et al., 2006). The MMPs acting through the EMT process could have direct and/or indirect role on the hESCs. The removal of physical barriers by the degradation of the extracellular matrix components and the migration of the cells during an EMT process could be allowed by the MMPs (Lee et al., 2006). MMPs also cleave non-matrix substrates such as growth factors, cytokines, chemokines, receptors and cell–cell adhesion molecules (McCawley and Matrisian, 2001). Several studies have demonstrated a direct degradation of cell adhesion molecules such as E-cadherin/catenin complexes by the MMPs (Polette et al., 2004; Lee et al., 2006). Moreover, it is known that integrin-matrix interactions play an important role during embryonic stem cell differentiation (Czyz and Wobus, 2001) and induce selective expression of MMPs (Bjorklund and Koivunen, 2005).
Taking together with the literature data, our results suggest that crucial interactions at the level of GSK-3 are implicated in the maintenance of hESCs pluripotency, the self-renewal and the EMT signalling pathways. Further experiments to study these complex interactions could help to improve culturing hESCs in feeder-free conditions with a defined medium.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Urielle Ullmann is a Research Assistant at the Fund for Scientific Research, Flanders (FWO-Vlaanderen). The work was supported by a grant from the FWO-Vlaanderen and the Research Council of the Vrije Universiteit Brussel.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
We gratefully acknowledge the assistance of Peter In't Veld from the Experimental Pathology Department of the Medical School of the VUB for the analyses of TEM pictures and Johan Schiettecatte from the Radioimmunology laboratory of the academic hospital of the VUB for ELISA tests and Dr Olivier Vandenberg from the Epidemiological and Statistical Department, Public Health School of the ULB (Université Libre de Bruxelles) for reviewing statistical analyses. The authors are also grateful to Walter Meul for computing pictures.
|
Footnotes |
|---|
This study was presented orally at the 23rd Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE) in Lyon (1–4 July 2007) (abstract O-166).
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
|---|
Bachelder RE, Yoon SO, Franci C, de Herreros AG, Mercurio AM. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition. J Cell Biol (2005) 168:29–33.
Barrallo-Gimeno A, Nieto MA. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development (2005) 132:3151–3161.
Behr R, Heneweer C, Viebahn C, Denker HW, Thie M. Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation. Stem Cells (2005) 23:805–816.
Biswas C, Zhang Y, Decastro R, Guo H, Nakamura T, Kataoka H, Nabeshima K. The human tumor cell derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res (1995) 55:434–439.
Björklund M, Koivunen E. Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta (2005) 1755:37–69.[Medline]
Cauffman G, Liebaers I, Van Steirteghem A, Van de Velde H. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells (2006) 24:2685–2691.
Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem (2003) 253:269–285.[CrossRef][Web of Science][Medline]
Ciruna B, Rossant J. FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak. Dev Cell (2001) 1:37–49.[CrossRef][Web of Science][Medline]
Czyz J, Wobus A. Embryonic stem cell differentiation: the role of extracellular factors. Differentiation (2001) 68:167–174.[CrossRef][Web of Science][Medline]
de Boer TP, van Veen TA, Bierhuizen MF, Kok B, Rook MB, Boonen KJ, Vos MA, Doevendans PA, de Bakker JM, van der Heyden MA. Connexin43 repression following epithelium-to-mesenchyme transition in embryonal carcinoma cells requires Snail1 transcription factor. Differentiation (2007) 75:208–218.[CrossRef][Web of Science][Medline]
Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci (2003) 116:1175–1186.
Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X, Cheng L. Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells (2005) 23:1489–1501.
Freije JM, Balbin M, Pendas AM, Sanchez LM, Puente XS, Lopez-Otin C. Matrix metalloproteinases and tumor progression. Adv Exp Med Biol (2003) 532:91–107.[Web of Science][Medline]
Fridman R, Toth M, Chvyrkova I, Meroueh SO, Mobashery S. Cell surface association of matrix metalloproteinase-9 (gelatinase B). Cancer Metastasis Rev (2003) 22:153–166.[CrossRef][Web of Science][Medline]
Gilles C, Polette M, Seiki M, Birembaut P, Thompson EW. Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Lab Invest (1997) 76:651–660.[Web of Science][Medline]
Gilles C, Polette M, Zahm JM, Tournier JM, Volders L, Foidart JM, Birembaut P. Vimentin contributes to human mammary epithelial cell migration. J Cell Sci (1999) 112:4615–4625.[Abstract]
Guarino M. Epithelial-to-mesenchymal change of differentiation. From embryogenetic mechanism to pathological patterns. Histol Histopathol (1995) 10:171–184.[Web of Science][Medline]
Hajra KM, Chen DY, Fearon ER. The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res (2002) 62:1613–1618.
Hay ED. The mesenchymal cell, its role in the embryo, and the remarkable signalling mechanisms that create it. Dev Dyn (2005) 233:706–720.[CrossRef][Web of Science][Medline]
Himelstein BP, Canete-Soler R, Bernhard EJ, Dilks DW, Muschel RJ. Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion Metastasis (1994) 14:246–258.[Web of Science][Medline]
Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T, Lako M. Downregulation of NANOG induces differentiation of embryonic stem cells to extraembryonic lineages. Stem Cells (2005) 23:1035–1043.
Igakura T, Kadomatsu K, Kaname T, Muramatsu H, Fan QW, Miyauchi T, Toyama Y, Kuno N, Yuasa S, Takahashi M, et al. A null mutation in basigin, an immunoglobulin superfamily member, indicates its important roles in peri-implantation development and spermatogenesis. Dev Biol (1998) 194:152–165.[CrossRef][Web of Science][Medline]
Itoh Y, Takamura A, Ito N, Maru Y, Sato H, S Suenaga N, Aoki T, Seiki M. Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J (2001) 20:4782–4793.[CrossRef][Web of Science][Medline]
Itoh Y, Seiki M. MT1-MMP: a potent modifier of pericellular microenvironment. J Cell Physiol (2006) 206:1–8.[CrossRef][Web of Science][Medline]
Katoh M, Katoh M. Cross-talk of WNT and FGF signalling pathways at GSK3beta to regulate beta-Catenin and SNAIL signalling cascades. Cancer Biol Ther (2006) 5:1059–1064.[Web of Science][Medline]
Kim K, Lu Z, Hay ED. Direct evidence for a role of beta-catenin/LEF-1 signalling pathway in induction of EMT. Cell Biol Int (2002) 26:463–476.[CrossRef][Web of Science][Medline]
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet (2005) 365:1636–1641.[CrossRef][Web of Science][Medline]
Lee JM, Dedhar S, Kallouri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol (2006) 172:973–981.
Lowry WE, Blanpain C, Nowak JA, Guasch G, Lewis L, Fuchs E. Defining the impact of beta-catenin/Tcf transactivation on epithelial stem cells. Genes Dev (2005) 19:1596–1611.
Ludwig TE, Levenstein ME, Jones JM, Berggen WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyck MS, et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotech (2006) 24:185–187.[CrossRef][Web of Science][Medline]
Maeda M, Johnson KR, Wheelock MJ. Cadherin switching: essential for behavioural but not morphological changes during an epithelium-to-mesenchyme transition. J Cell Sci (2005) 118:873–887.
Mannello F, Tonti GA, Papa S. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells. Stem Cells (2006) 24:475–481.
Mateizel I, De Temmerman N, Ullmann U, Cauffman G, Sermon K, Van de Velde H, De Rycke M, Degreef E, Devroey P, Liebaers I, et al. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod (2006) 21:503–511.
McCawley LJ, Matrisian LM. Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol (2001) 13:534–540.[CrossRef][Web of Science][Medline]
Miyabayashi T, Teo JL, Yamamoto M, McMillan M, Nguyen C, Kahn M. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci USA (2007) 104:5668–5673.
Muller T, Bain G, Wang X, Papkoff J. Regulation of epithelial cell migration and tumor formation by beta-catenin signalling. Exp Cell Res (2002) 280:119–133.[CrossRef][Web of Science][Medline]
Nawrocki-Raby B, Gilles C, Polette M, Martinella-Catusse C, Bonnet N, Puchelle E, Foidart JM, Van Roy F, Birembaut P. E-Cadherin mediates MMP down-regulation in highly invasive bronchial tumor cells. Am J Pathol (2003) 163:653–661.
Nieto MA, Sargent MG, Wilkinson DG, Cooke J. Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science (1994) 264:835–839.
Peinado H, Portillo F, Cano A. Switching on-off Snail: LOXL2 versus GSK3beta. Cell Cycle (2005) 4:1749–1752.[Web of Science][Medline]
Polette M, Nawrocki-Raby B, Gilles C, Clavel C, Birembaut P. Tumor invasion and matrix metalloproteinases. Crit Rev Oncol Hematol (2004) 49:179–186.[Web of Science][Medline]
Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, Carpenter MK. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn (2004) 229:259–274.[CrossRef][Web of Science][Medline]
Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature (1994) 370:61–65.[CrossRef][Medline]
Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med (2004) 10:55–63.[CrossRef][Web of Science][Medline]
Sato H, Takino T, Miyamori H. Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis. Cancer Sci (2005) 96:212–217.[CrossRef][Medline]
Spencer HL, Eastham AM, Merry CL, Southgate TD, Perez-Campo F, Soncin F, Ritson S, Kemler R, Stern PL, Ward CM. E-cadherin inhibits cell surface localization of the pro-migratory 5T4 oncofetal antigen in mouse embryonic stem cells. Mol Biol Cell (2007) 18:2838–2851.
Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T, Stojkovic M. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells (2005) 23:306–314.
Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol (2006) 7:131–142.[CrossRef][Web of Science][Medline]
Tseng AS, Engel FB, Keating MT. The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem Biol (2006) 13:957–963.[CrossRef][Web of Science][Medline]
Ullmann U, In't Veld P, Gilles C, Sermon K, De Rycke M, Van de Velde H, Van Steirteghem A, Liebaers I. Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Mol Hum Reprod (2007) 13:21–32.
Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev (2000) 14:2123–2133.
Wang H, Wen Y, Mooney S, Li H, Behr B, Polan ML. Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human preimplantation embryos. Fertil Steril (2003) 80:736–742.[CrossRef][Web of Science][Medline]
Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature (2003) 423:448–452.[CrossRef][Medline]
Wong RC, Pebay A, Nguyen LT, Koh KL, Pera MF. Presence of functional gap junctions in human embryonic stem cells. Stem Cells (2004) 22:883–889.
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol (2001) 19:971–974.[CrossRef][Web of Science][Medline]
Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells (2005) a23:315–323.
Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods (2005) b2:185–190.[CrossRef][Web of Science][Medline]
Zhou BP, Hung MC. Wnt, hedgehog and snail: sister pathways that control by GSK-3beta and beta-Trcp in the regulation of metastasis. Cell Cycle (2005) 4:772–776.[Web of Science][Medline]
Zhou Z, Apte SS, Soininen R, Cao R, Baaklini GY, Rauser RW, Wang J, Cao Y, Tryggvason K. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci USA (2000) 97:4052–4057.
Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, Hung MC. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol (2004) 10:931–940.
Zucker S, Hymowitz M, Rollo EE, Mann R, Conner CE, Cao J, Foda HD, Tompkins DC, Toole BP. Tumoregenic potential of extracellular matrix metalloproteinase inducer. Am J Pathol (2001) 158:1921–1928.
Submitted on July 31, 2007; resubmitted on December 28, 2007; accepted on January 3, 2008.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||