title: Dimeric integrin alpha5beta1 ligands confer morphological and differentiation responses to murine embryonic stem cells.
abstract: We present the first report utilizing, and showing the functional relevance of, self-assembling polyvalent ligands specific for integrin alpha5beta1 in murine embryonic stem (mES) cell adhesion. Di, tri and tetrameric 9th-10th type III fibronectin domains (FIII9'10) were used to generate clustered integrin alpha5beta1 ligand surfaces for mES cell culture. Compared to gelatin, FIII9'10 (monomer), FIII9'10-trimer and -tetramer, the FIII9'10-dimer supported the highest number of mES cell colonies. No evidence of domain unfolding upon surface adsorption was found. Colonies appeared disperse with a spread cell morphology unless subdued back to a tight morphology with increasing concentrations of leukemia inhibitory factor (LIF). In the presence of LIF, mES cells adherent to the FIII9'10-dimer showed transient upregulation of Oct-4, the mesodermal transcription factor, Brachyury, and the ectodermal marker, Nestin. However, dual upregulation of Nanog maintained the mES cells in a pluripotent state, confirmed by alkaline phosphatase staining. Therefore, the behavior of mES cells adherent to dimeric integrin alpha5beta1 ligands is a largely morphological phenomenon conferring pro-differentiation signals towards mesodermal and ectodermal lineages. This work will be of interest to cell and tissue engineering groups aiming to control ES cell behavior through integrin ligand presentation and synthetic substrates.
authors.affiliation: Division of Immunology Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Singh
authors.forename: Michaela
authors.initials: M
authors.lastname: Kreiner
authors.forename: Clive S
authors.initials: CS
authors.lastname: McKimmie
authors.forename: Stephen
authors.initials: S
authors.lastname: Holt
authors.forename: Christopher F
authors.initials: CF
authors.lastname: van der Walle
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Graham
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D005353:Fibronectins / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D008024:Ligands; D051379:Mice; D055503:Protein Multimerization; D023822:Tissue Engineering; D014158:Transcription, Genetic
chemical_list: D005353:Fibronectins; D039081:Integrin alpha5beta1; D053605:Leukemia Inhibitory Factor; D008024:Ligands