Fibronectin-mediated upregulation of alpha 5 beta 1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that alpha 5 beta 1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specifically mediated by integrin alpha 5 beta 1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective upregulation of the alpha 5 beta 1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, alpha-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of alpha 5 beta 1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of alpha 5 beta 1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.