Although a number of defined media have been described that support pluripotent stem cell growth, most rely on the use of culture dishes coated with substrates that are relatively impure preparations of extracellular matrices. This report describes the use of highly pure recombinant hE-cad-Fc as a substitute for basement membrane that facilitates maintenance of pluripotency under completely defined conditions. Pluripotent stem cells cultured on an hE-cad-Fc-coated surface were virtually indistinguishable from those grown on a Matrigel-coated surface, including cell morphology, rate of proliferation, maintenance of an undifferentiated phenotype, and ability to differentiate into multiple cell types in both embryoid bodies and teratomas. The one difference was noted when cells were passaged using protease digestion cocktails such as Accutase, in that their adhesion efficiency was reduced; however, reduced adherence was circumvented by using enzyme-free Cell Dissociation Buffer to passage the cells. Indeed we believe that the use of enzyme-free methods to passage pluripotent stem cells has advantages, including the preservation of cell-surface proteins that can be used for FACS analyses and sorting.
Historically, integrin-mediated cell-ECM interactions have been considered essential for maintenance of stem cell pluripotency and viability [24
]. Based on such studies, a significant effort has been devoted to finding a suitable ECM component that can maintain interactions with these cell surface receptors. In mouse ES cells, type I and IV collagen have been reported as contributing to the maintenance of pluripotency [27
], although mouse ES cells don't appear to express α1ß1 or α2ß1 integrins, which are the predominant receptors through which cells adhere to collagen within a basement membrane. In addition, laminin-511 was also found to enable the maintenance of pluripotency of mouse ES cells through interactions with α6ß1 and α5ß1 integrins [28
]. Recently recombinant vitronectin [29
], collagen [30
] and laminin-511 [31
] have been reported as matrices that support human ES cells pluripotency all of which facilitate binding through interaction with integrins.
The absence of integrin binding proteins within the hE-cad-Fc substratum may indicate that integrin-mediated signalling is dispensable for stem cell maintenance. Integrin-ECM interactions predominantly act through integrin-linked kinase (ILK) or focal adhesion kinase (FAK) signalling pathways, and a subset of integrins have been shown to activate PI3K/Akt and MAPK pathways [32
]. Although E-cadherin-mediated adhesion is commonly associated with ß-catenin signalling, it also stimulates PI3K/Akt signalling at least in ovarian carcinoma cells [33
]. The Akt signalling pathway has been considered important for maintenance of pluripotency in mouse, primate, and human ES cells [34
]. Although further analyses are required before the exact mechanism through which the hE-cad-Fc surface can support pluripotency is fully understood, we speculate that trans-homodimerization between cell surface E-cadherin and the E-cadherin domain presented through the hE-cad-Fc surface could facilitate stem cell maintenance by activation of PI3K/Akt signalling pathway. Consistent with this reasoning, several studies have implicated a role for E-cadherin in controlling both pluripotency and differentiation of ES cells [15
], although the mechanism through which E-cadherin regulates these processes appears to be complex.
We previously reported that using mouse E-cad-Fc as a substratum, mouse ES cells, although retaining pluripotency, transition to a mesenchymal morphology and scatter across the plate rather than forming colonies [19
]. In contrast, human pluripotent stem cells retain their ability to form colonies and show no evidence of undergoing an epithelial to mesenchymal transition (Figures and ). The mechanisms underlying the different response to culture on E-cadherin substrate between mouse and human pluripotent stem cells have yet to be defined. However, human and mouse embryonic stem cells have been shown to display substantial differences in expression of transcription factors, cytokines, cell surface markers, growth factor receptors, and proteins involved in cell cycle control [37
]. These differences in gene expression between mouse and human ES cells can often explain functional distinctions between the cell types, including the independence of human ES cells on LIF for maintaining pluripotency, the ability of human but not mouse ES cells to form trophectoderm in culture, and the relatively slow rate of proliferation of human ES cells compared to that of mouse. Harb et al
. demonstrated that the inhibition of Rho-ROCK pathway induces cell scattering of human ES cells [38
], indicating that there are different mechanisms that control cytoskeletal reorganization in human and mouse ES cells. Although both mouse and human ES cells express E-cadherin, it seems possible that a subset of the myriad of proteins that interact to define E-cadherin biological activity could differ between mouse and human ES cells, and experiments to address this are currently underway.