RESUMO
The maintenance of the pluripotency of human embryonic stem (hES) cells requires special conditions for culturing. These conditions include specific growth factors containing media and extracellular matrix (ECM) or an appropriate substrate for adhesion. Interactions between the cells and ECM are mediated by integrins, which interact with the components of ECM in active conformation. This study focused on the characterisation of the role of integrin ß1 in the adhesion, migration and differentiation of hES cells. Blocking integrin ß1 abolished the adhesion of hES cells, decreasing their survival and pluripotency. This effect was in part rescued by the inhibition of RhoA signalling with Y-27632. The presence of Y-27632 increased the migration of hES cells and supported their differentiation into embryoid bodies. The differences in integrin ß1 recycling in the phosphorylation of the myosin light chain and in the localisation of TSC2 were observed between the hES cells growing as a single-cell culture and in a colony. The hES cells at the centre and borders of the colony were found to have differences in their morphology, migration and signalling network activity. We concluded that the availability of integrin ß1 was essential for the contraction, migration and differentiation ability of hES cells.
RESUMO
The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore, understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study, we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES) cells (H9 cell line). Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise, expression of ectodermal (SOX1 and OTX2) and endodermal (GATA4 and FOXA2) markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133, we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion, lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential.