The niche in which stem cells reside is involved in the regulation of stem cell fate, such as differentiation and self-renewal, by providing ECM proteins, growth factors, cell-cell interactions and balancing chemical factors such as the level of oxygen and pH. ECM proteins are involved in maintaining stemness of stem cells and in regulating differentiation via integrin-mediated signalling. Following the interaction of ECM proteins with integrins, integrins cluster and interact with large complexes of signalling proteins. These adhesion complexes have been reported to contain at least 150 proteins, which have been termed the adhesome. Adhesion complex proteins interact with the actin cytoskeleton and signalling pathways to play an essential role in stem cell fate. The hypothesis in this study was that the interaction of stem cells via integrin receptors with ECM proteins, lead to changes in the abundance or composition of adhesion complexes, which potentially activates signalling pathways involved in either maintaining or differentiation of stem cells. In this study, three principal advances have been made:First, a method was developed using ligand-coated magnetic beads for the isolation of integrin-associated complexes from pluripotent human embryonic stem cells (hESCs). The isolated integrin-associated complexes from hESCs were analysed by proteomic methods, which led to the detection of key integrin-associated adhesion proteins such as talin, vinculin, alpha actinin 4, filamin B, filamin C and zyxin. Second, isolation of integrin-associated complexes from multipotent MSCs was performed using a method based on “de-roofing” MSCs from FN or PDL coated plastic dishes, leading to the detection of key adhesome components by mass spectrometry. Ontological analysis of proteins enriched on FN demonstrated the enrichment of adhesion complexes. Third, following the induction of multipotent MSCs into early adipogenic MSCs and the isolation of integrin-associated complexes from early adipogenic MSCs and undifferentiated MSCs, core adhesome components were identified in induced and non-induced MSCs, with induction hypothesised to cause putative changes in the FN-induced adhesome network. Also, the level of adhesion complexes increased significantly in MSCs on FN upon induction into adipocytes compared to non-induced MSCs on FN and versus the control as shown by bioinformatics analysis. This data led to the hypothesis that upon induction of MSCs into adipocytes the abundance of proteins in integrin-associated complexes or the number of adhesion complexes increases.In conclusion, in this study two biochemical affinity methods were developed for the isolation of integrin-associated complexes from hESCs and MSCs, using ligand coated magnetic beads and ligand-coated plastic dishes. The development of these methods led to the isolation of adhesion-related proteins from pluripotent hESCs and differentiated MSCs and the detection of a pattern of changes in the abundance of adhesion related proteins in differentiated MSCs incubated on FN. The development of methods for the isolation of adhesion related complexes from stem cells can lead to a better understanding of the role of adhesion in differentiation and maintenance of pluripotency in stem cells. A better understanding of adhesion could have future implications in obtaining pure populations of undifferentiated stem cells for cell-based therapies and differentiated cells for the use in tissue engineering and repair.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:564355 |
| Date | January 2012 |
| Creators | Ajeian, Jila |
| Contributors | Kimber, Susan; Humphries, Martin |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/proteomic-analysis-of-integrinassociated-complexes-from-stem-cells(c632bcd0-d997-4a26-9891-d3486ecb241a).html |
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