The epithelial–mesenchymal transition (EMT) in equine amniotic multipotent progenitor cells induces changes of the cell glycan profile

ABSTRACT - The glycoprotein pattern was investigated in equine amniotic epithelial, mesenchymal and transdifferentiated cells with a panel of twelve lectins, in combination with saponification and sialidase digestion (K-s). NeuNacα2,3Galβ1,4GlcNAc, NeuNAcα2,6Gal/GalNAc and GalNAc (MAL II, SNA, SBA affinity) were mainly expressed in the epithelial cells, less in the mesenchymal cells and even lesser in the transdifferentiated ones, whereas N-linked glycans from high-Man type glycans (Con A reactivity) showed an opposite trend. Fucosylated LTA-binding glycans were present in epithelial and mesenchymal cells but not in the transdifferentiated cells which, on the contrary, contained terminal L-Fucα1,2Galβ1,4GlcNAcβ and αGal residues (UEA I and GSA I-B4 binding). Interestingly, only transdifferentiated cells expressed NeuNacGalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 and Galβ1,3GalNAc terminating glycans (K-s-DBA and PNA affinity). These findings show that the epithelial–mesenchymal transition (EMT) in equine amniotic multipotent progenitor cells is characterized by glycosylation changes which produce cell specific glycan profile. INTRODUCTION - The epithelial to mesenchymal transition (EMT) is the process by which epithelial cells dramatically alter their shape and motile behavior as they differentiate into mesenchymal cells. During the EMT, epithelial cells lose their apical-basal polarity and extensive adhesions to neighboring cells and basement membranes. Coincident with the modification of cell-cell and cell substrate adhesions, the transitioning epithelial cell undergoes a dramatic shape change, adopting an extensively flattened and elongated leading-trailing mesenchymal morphology (Mendez et al., 2010). The EMT and the reverse process, termed the mesenchymal-epithelial transition (MET), play central roles in embryogenesis (Thiery and Sleeman, 2006). For example, in the embryo itself, the first EMT event occurs at gastrulation. A subset of cells from the epiblast, the single epithelial cell layer of the embryo, move to the midline to form the primitive streak, a linear structure that bisects the embryo along the antero-posterior axis. These cells undergo EMT and internalize to generate mesoderm and endoderm, while those remaining in the epiblast become ectoderm. Thus, the embryo is transformed from a single layer to three germ layers. Mesoderm and endoderm contribute to many tissues of the adult organism by undergoing several rounds of EMT and MET. Primary EMT also occurs during vertebrate nervous system development to generate neural crest cells. The epithelial neural plate in the midline of the embryo rolls up to form the cylindrical neural tube, precursor to the brain and spinal cord. Those epithelial cells located near the dorsal midline of the neural tube undergo primary EMT to become migratory neural crest cells, which subsequently move away from the neural tube, navigating along stereotypic pathways throughout the embryo. (Le Douarin and Kalcheim, 1999). Primary EMT events such as gastrulation and formation of migratory neural crest cells are followed by the generation of distinct cell types. Gastrulation and neural crest formation are two prototypical processes governed by EMT in amniotes. But, EMT-like process is also evoked during tumor progression and metastasis emergence where, as in embryogenesis, cells undergo EMT to migrate and colonize distant territories. While EMT and its converse MET, are concepts first defined by Elizabeth Hay about 40 years ago (Hay et al. 1968), the role of EMT-like process during tumor progression is usually not well understood. It is noteworthy that in placental mammals the epithelial layer of amnion originates from the trophectoderm and it is continuous with the epiblast, then it is reasonable to speculate that some amniotic epithelial cells may escape the specification that accompanies gastrulation, and may retain some (or all) of the characteristics of epiblastic cells, such as pluripotency, behaving as stem cells that are able to preserve intrinsically the ability to transdifferentiate. Since it seems very likely that malignant cells use the same mechanisms during the formation of metastasis in vivo, the amniotic mesenchymal cells (AMCs) could represent a good model to study this phenomenon in vitro that we observed to occur spontaneously in our AMCs culture conditions. Glycoproteins are important determinants for many biological activities and undergo molecular changes during cell differentiation as well as from normal to pathological conditions (Varki, 1999). Lectins are a family of carbohydrate-binding proteins that recognize and distinguish specific sugar structures and have been extensively used to identify, characterize, and isolate novel cell subpopulations. For example, the lectin Dolichos biflorus agglutinin (DBA), which recognizes α-Nacetylgalactosamine (αGalNAc), was used to characterize mouse embryonic stem cell cultures (Nash et al., 2007). Lectins have also been used to investigate metastatic processes in many cancer types (Plattner et al., 2008), as well as to document the repertoire of glycoepitopes on the surface of embryonic carcinoma cells (Muramatsu et al., 1985). Differentiated progeny of mesenchymal stem cells (MSCs) differ in N-linked glycans that impact myelopoietic supportive capacity (Morad et al., 2008). These findings indicate that glycans can be used as markers to define specific stages of stemness in multiple cell types. The aim of this study was to characterize the glycoprotein pattern expressed in the glycocalyx from equine amniotic epithelial, mesenchymal and transdifferentiated cells by means of the lectin histochemistry. MATERIALS AND METHODS - Tissue collection. Three amniotic membranes (AM) were obtained at term of normal pregnancies and after vaginal delivery from three mares. Cell isolation and culture. Amniotic derived cells were isolated and cultured as reported by Lange-Consiglio et al. (2012). The study was performed at passage (P) 3 on epithelial and mesenchymal cells while at P1 and P2 for transdifferentiated cells. Cryopreservation. Cells were frozen at P3 in HG-DMEM supplemented with 50% FBS and 10% DMSO for 1 months at -196°C in liquid nitrogen. Glycoprofiling. After thawing, the glycoanalysis of the cultured cells was performed with a panel of twelve lectins (MAL II, SNA, PNA, RCA120, GSA I-B4, DBA, HPA,SBA, Con A, GSA II, UEA I, LTA), in combination with saponification and sialidase digestion (K-s), according to Desantis et al. (2010). RESULTS – Cell culture. Isolated cells readily attach to plastic culture dishes. Amniotic epithelial cells display typical polygonal epithelial morphology, while amniotic mesenchymal cells are fibroblast-like. Sometime, during in vitro culture, epithelial cells lose their polygonal morphology and undergo a shape change, adopting an extensively elongated mesenchymal morphology. Glycosylation pattern. Lectin histochemistry revealed: 1) gradual decreasing of MAL II,SNA, SBA binders but a progressive increase of Con A reactivity in epithelial, mesenchymal and transdifferentiated cells; 2) fucosylated LTA binding glycans in epithelial and mesenchymal cells; 3) terminally fucosylated and/or galactosylated glycans (UEA I and GSA I-B4 affinity) in P1 and P2 transdifferenziated cells; 4) specific PNA and K-s-DBA binding sites in P1 transdifferentiated cells. DISCUSSION - Recently, for the first time in equine species, stem cells from extra-fetal tissue, such as amnion, have been retrieved (Lange-Consiglio et al., 2012). Amnion-derived mesenchymal stem cells (AMCs) share specific characteristics with embryonic and adult stem cells, expressing representative mesenchymal (CD105, CD44, CD29, CD166) and pluripotent (Tra-1-60, SSEA-4, Oct-4) markers, being highly proliferative and retaining high plasticity, as supported by their capacity to differentiate into multiple germ layers (mesoderm and ectoderm). We also provided evidences to support that when allogeneically transplanted in vivo AMCs are well tolerated and exert beneficial effects on tendon regeneration after spontaneous lesions better than adult bone marrow mesenchymal stem cells (Lange Consiglio et al. 2013). This in vivo therapy confirm the potential application of these cells in regenerative medicine, indeed, the clinical and ultrasonographical evaluation of tendons, after two years of follow-up, did not reveal evidences of inappropriate tissue or tumor formation and the clinical outcomes after MSCs transplantation are favorable with an obvious active proliferative healing in the area injected with AMSCs. These important data exclude the involvement of these cells in tumor processes but the spontaneous epithelial-mesenchymal transition during in vitro culture make them interesting to study this spontaneous phenomenon as model in tumorigenic process. Therefore, we studied their glycocalyx composition because the glycosylation pattern is involved in the cell differentiation and behaviour (Varki et al., 1999). We used a panel of twelve lectins among the more frequently used ones in order to detect the presence of the six sugars (D-mannose, L-fucose, D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and neuraminic acid) constituting the animal oligosaccharides, to characterize their sequence in the oligosaccharide chains of the glycoproteins and to distinguish the two main categories of glycoproteins: O-linked and N-linked. The results suggests that O-linked glycans terminating with NeuNacα2,3Galβ1,4GlcNAc and/or NeuNAcα2,6Gal/GalNAc and/or GalNAc are mainly expressed in the epithelial cells, less in the mesenchymal cells and even lesser in the transdifferentiated ones, whereas N-linked glycans from high-Man type glycans show an opposite trend. In addition, N-linked fucosylated LTA-binding glycans are present in epithelial and mesenchymal cells but not in the transdifferentiated cells which, on the contrary, contain terminal L-Fucα1,2Galβ1,4GlcNAcβ and αGal residues. Interestingly, P1 but not P2 transdifferentiated cells express O-linked glycans terminating with NeuNacGalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 and Galβ1,3GalNAc. Although, it is not possible to establish the exact function of the detected glycans, because no previous studies have been performed on these cells, this study demonstrates that the epithelial–mesenchymal transition (EMT) in equine amniotic multipotent progenitor cells induces changes in cell surface glycan profile that could be responsible of the alteration of their shape and motile behavior. REFERENCES – 1. Hay E.D. Organization and fine structure of epithelium and mesenchyme in the developing chick embryo. In Epithelial-mesenchymal interactions. R. Fleischmajer and R.E. Billingham, editors. Williams & Wilkins. Baltimore, Maryland, USA. 31-55 (1968). 2. Lange-Consiglio A. et al. Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane. 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