Abstract
Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development by which sessile epithelial cells are converted into migratory mesenchymal cells. Our laboratory has been successful in the establishment of Xenopus tropicalis immature Sertoli cells (XtiSCs) with the restricted differentiation potential. The aim of this study is the determination of factors responsible for EMT activation in XtiSCs and stemness window acquisition where cells possess the broadest differentiation potential. For this purpose, we tested three potent EMT inducers—GSK-3 inhibitor (CHIR99021), FGF2, and/or TGF-β1 ligand. XtiSCs underwent full EMT after 3-day treatment with CHIR99021 and partial EMT with FGF2 but not with TGF-β1. The morphological change of CHIR-treated XtiSCs to the typical spindle-like cell shape was associated with the upregulation of mesenchymal markers and the downregulation of epithelial markers. Moreover, only CHIR-treated XtiSCs were able to differentiate into chondrocytes in vitro and cardiomyocytes in vivo. Interestingly, EMT-shifted cells could migrate towards cancer cells (HeLa) in vitro and to the injury site in vivo. The results provide a better understanding of signaling pathways underlying the generation of testis-derived stem cells.
Highlights
Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), occur in numerous developmental processes including mesoderm, neural crest, and testicular formations [1]
To induce epithelialmesenchymal transition (EMT), cells were cultured in a growth medium overnight before its replacement by induction medium supplemented with CHIR99021 (CHIR; GSK-3 inhibitor, 3 μM, Sigma), mFGF2 (25 ng/ml), or hTGF-β1 (2.5, 5, or 10 ng/ml) for 3-4 days
To test the expression of two IFs, cytokeratin and vimentin, in X. tropicalis Sertoli cell (SC), testicular sections from juvenile (6-month-old) and adult (3-year-old) X. tropicalis were employed for double staining with two antibodies against cytokeratin/Sox9 or vimentin/Sox9 (Sox9, a specific marker of SCs)
Summary
Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), occur in numerous developmental processes including mesoderm, neural crest, and testicular formations [1]. EMT ensures both, high migratory capacity and the cell stemness. Several groups have tried to generate mesenchymal stem cells (MSCs) by inducing EMT in cultured human epithelial cells. The immortalized human mammary epithelial cells were incubated with recombinant TGF-β1 or transduced with vectors expressing EMT key transcription factors, Snai or twist. Treated cells have exhibited the characteristics of MSCs, including specific antigenic profile, the capacity to differentiate into mesodermal cell types and to migrate towards cancer cells and wound injury sites [7, 8]
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