A Regulatory Network Involving ß-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self-renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two-layer regulatory circuit involving ß-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of ß-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on the cell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Akt signaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular ß-catenin binding domain induces TCF-eGFP activity and promotes hESC differentiation through ß-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a ß-catenin "sink" sequestering free cytoplasmic ß-catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self-renewal associated with short-term Wnt/ß-catenin activation to definitive differentiation. Stem Cells 2015;33:1419-1433.

A unique interplay between Rap1 and E-cadherin in the endocytic pathway regulates self-renewal of human embryonic stem cells.

Regulatory mechanisms pertaining to the self-renewal of stem cells remain incompletely understood. Here, we show that functional interactions between small GTPase Rap1 and the adhesion molecule E-cadherin uniquely regulate the self-renewal of human embryonic stem cells (hESCs). Inhibition of Rap1 suppresses colony formation and self-renewal of hESCs, whereas overexpression of Rap1 augments hESC clonogenicity. Rap1 does not directly influence the expression of the pluripotency genes Oct4 and Nanog. Instead, it affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell-cell cohesion, which is essential for the colony formation and self-renewal of hESCs. Conversely, distinct from epithelial cells, disruption of E-cadherin mediated cell-cell adhesions induces lysosome delivery and degradation of Rap1. This in turn leads to a further downregulation of E-cadherin function and a subsequent reduction in hESC clonogenic capacity. These findings provide the first demonstration that the interplay between Rap1 and E-cadherin along the endocytic recycling pathway serves as a timely and efficient mechanism to regulate hESC self-renewal. Given the availability of specific activators for Rap1, this work provides a new perspective to enable better maintenance of human pluripotent stem cells.

Individual cell movement, asymmetric colony expansion, rho-associated kinase, and E-cadherin impact the clonogenicity of human embryonic stem cells.

Clonality is, at present, the only means by which the self-renewal potential of a given stem cell can be determined. To assess the clonality of human embryonic stem cells (hESC), a protocol involving seeding wells at low cell densities is commonly used to surmount poor cloning efficiencies. However, factors influencing the accuracy of such an assay have not been fully elucidated. Using clonogenic assays together with time-lapse microscopy, numerical analyses, and regulated gene expression strategies, we found that individual and collective cell movements are inherent properties of hESCs and that they markedly impact the accuracy of clonogenic assays. Analyses of cell motility using mean-square displacement and paired migration correlation indicated that cell movements become more straight-line or ballistic and less random-walk as separation distance decreases. Such motility-induced reaggregation (rather than a true clone) occurs approximately 70% of the time if the distance between two hESCs is <6.4 mum, and is not observed if the distance is >150 mum. Furthermore, newly formed small hESC colonies have a predisposition toward the formation of larger colonies through asymmetric colony expansion and movement, which would not accurately reflect self-renewal and proliferative activity of a true hESC clone. Notably, inhibition of Rho-associated kinase markedly upregulated hESC migration and reaggregation, producing considerable numbers of false-positive colonies. Conversely, E-cadherin upregulation significantly augmented hESC clonogenicity via improved survival of single hESCs without influencing cell motility. This work reveals that individual cell movement, asymmetric colony expansion, Rho-associated kinase, and E-cadherin all work together to influence hESC clonogenicity, and provides additional guidance for improvement of clonogenic assays in the analysis of hESC self-renewal.