RESUMEN
Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine, leukaemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, and this is accompanied by an increase in cell-substratum adhesion and cell spreading. The purpose of this study was to investigate the relationship between cell spreading and mESC differentiation. Using E14 and R1 mESC lines, we have restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biomaterial substrates, or by manipulating the cytoskeleton. We demonstrate that by restricting the degree of spreading by either method, mESCs can be maintained in an undifferentiated and pluripotent state. Under these conditions, self-renewal occurs without the need for LIF and is independent of nuclear translocation of tyrosine-phosphorylated STAT3 or ß-catenin, which have previously been implicated in self-renewal. We also demonstrate that the effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as evidenced by the observations that inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or siRNA do not promote differentiation. These results show that mESC spreading and differentiation are regulated both by LIF and by cell-substratum adhesion, consistent with the hypothesis that cell spreading is the common intermediate step in the regulation of mESC differentiation by either LIF or cell-substratum adhesion.
Asunto(s)
Materiales Biocompatibles/farmacología , Movimiento Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/enzimología , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Factor Inhibidor de Leucemia/farmacología , Ratones , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Plasma polymer surfaces were fabricated such that the cell response to a range of carboxylic acid concentrations on a single sample could be investigated. Surface chemical gradients from hydrophobic plasma polymerised octadiene (OD) to a more hydrophilic plasma polymerised acrylic acid (AA) were formed on glass coverslips. Surface characterisation of the chemical gradients was performed using X-ray photoelectron spectroscopy to determine elemental composition. Following culture of E14 and R1 mouse embryonic stem cells (mES) in differing culture media, cell pluripotency was determined by alkaline phosphatase staining. The results demonstrate that for these cell lines the capacity for self-renewal is maintained if the cells are restricted in their spreading to <120 microm2.