Regulation of differentiation and the maintenance of pluripotency in embryonic stem cells by beta-catenin.

摘要

Stem cells are cells capable of giving rise to differentiated daughter cells or to daughter cells that are identical, in terms of the cell's pluripotency, to the mother cell. The mechanism by which stem cells maintain themselves in a pluripotent state is presumably mediated by molecules with "stemness" activity. The work presented in this thesis attempts to understand how embryonic stem (ES) cells decide to differentiate into the neural lineage or to remain in a pluripotent state. The focus of this thesis is beta-catenin, a molecule involved in multiple cellular processes, including cell proliferation, stemness, and neuronal differentiation. We find that neuronal differentiation of ES cells is inhibited by differentiation at high density even during retinoic acid (RA) inductions. During high density differentiation, beta-catenin is preferentially localized to the cell membrane and is NH2-terminally phosphorylated. The net effect of this is decreased beta-catenin signaling during culture at high density. Elevation of beta-catenin signaling in high density cultures by treatment with wnt3a conditioned media, expression of a dominant negative form of E-cadherin, or artificial elevation of beta-catenin by expression of beta-catenin under the control of a constitutively active CMV promoter permits neuronal differentiation at high density. The central armadillo domain of beta-catenin was found to be necessary for neuronal differentiation whereas the C-terminal domain was not required. Expression of beta-catenin permitted neuronal differentiation even in the absence of RA, although RA treatment potentiated the effect. Activation of beta-catenin signaling did not affect proliferation of ES cells cultured with LIF. On the other hand, neuronal differentiation of beta-catenin expressing ES cells by LIF withdrawal was accompanied by decreased proliferation relative to the empty vector treated controls.; Human embryonic stem (HES) cells require growth on mitotically inactivated mouse embryonic feeder cells (MEFs) in order to be maintained in a pluripotent state. MEF plasma membrane preps were found to possess beta-catenin stabilizing activity. Treatment with a frizzled receptor body or treatment with beta1-integrin blocking antibodies did not block the MEF plasma membrane associated protein effect. HES cells were found to have beta-catenin diffusely localized throughout the cells, a finding indicative of activated beta-catenin. (Abstract shortened by UMI.)