Embryonic stem (ES) cells have the capacity to form any cell type. However, their propagation and differentiation is limited by current two dimensional (2D) culture techniques which offer little flexibility in terms of surface structure and functionalisation with bioactive molecules. The aim of the current work was to produce a novel scaffold that could manipulate ES cell behaviour using both architectural and biological cues. Electrospinning is a flexible technique that creates nonwoven meshes that mimic the fibrous architecture of the ECM. Initial work focused on investigating the suitability of electrospun poly(lactic-co-glycolic acid) (PLGA) meshes for 2D and three dimensional (3D) culture of mouse ES cells, with the hypothesis that the fibrous architecture would assist in maintaining pluripotency. The study also sought to functionalise the scaffolds with biologically active molecules. Heparan sulphate proteoglycans (HSPGs) reside at the cell surface and within the ECM where they mediate growth factor binding, assist cell attachment and stabilise the ECM. Furthermore, ES cells modulate their own microenvironment by controlling the composition of heparan sulphate (HS), regulating the binding of growth factors such as fibroblast growth factor (FGF) family members. Therefore, we aimed to immobilise HS and heparin (a highly sulphated structural analogue of HS) on the fibre surface in a form that was freely accessible for protein/cell interactions and that retained its biological activity. Electrospinning parameters were optimised to produce microfibre electrospun meshes with an average fibre diameter of 570nm. Cell morphology, proliferation and pluripotency were monitored using an Oct4-GFP reporter cell line and results compared with flat spin coated films. To investigate the potential for 3D culture, spinning parameters were altered to increase fibre diameter to >3micro metre with infiltration assessed using pro-migratory E-cadherin-/- ES cells. Scaffolds were coated with plasma polymerised allylamine (ppAm) to enable non-covalent immobilisation of HS/heparin. Ligand binding assays with the link module of TSG-6 and anti-heparin/HS antibodies were used to probe HS/heparin presentation on the fibre surface. The biological activity of the immobilised HS/heparin was analysed by testing the ability of coated scaffolds to rescue the neural differentiation capacity HS deficient EXT1-/- ES cells. Finally, human ES cells were cultured on the surface of ppAm scaffolds +/- HS in both unconditioned and mouse embryonic fibroblast (MEF) conditioned media for 5 days. Both microfibre meshes and flat spin coated films supported the attachment, growth and pluripotency of mouse ES cells. Cells adopted distinct morphologies, with mouse ES cells aggregating in rounded colonies on microfibre scaffolds and demonstrating increased spreading on spin coated films. Fibres >3micro metre created a thicker mesh with potential for 3D culture supporting the infiltration of E-cadherin-/- ES cells. ppAm enabled non-covalent immobilisation of HS/heparin in a form that was free to participate in protein interactions and which presented essential sulphation motifs within the HS/heparin chains. Bound HS was biologically active and functioned in synchrony with FGF4 to enhance neural differentiation of EXT1-/- ES cells. The constructs also supported the attachment and growth of human ES cells, with HS functionalised scaffolds demonstrating a slight increase in compatibility during culture in unconditioned media. The successful functionalisation of electrospun meshes with HS/heparin creates a highly versatile scaffold for ES cell culture and differentiation. The architecture of the meshes can be manipulated to either serve as a fibrous substrate for maintenance of pluripotency or support the formation of complex cell interactions present in vivo. The immobilisation of HS provides an extra dimension of versatility, as the scaffold can be tailored with specific HS species, potentially enabling the differential regulation of growth factor binding.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:713530 |
| Date | January 2010 |
| Creators | Meade, Kate |
| Contributors | Merry, Catherine |
| 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/glycosaminoglycan-gag-functionalised-electrospun-polylacticcoglycolic-acid-plga-scafffolds-for-the-propagation-and-differentiation-of-mouse-and-human-embryonic-stem-cells(fe5a59a7-46e0-4370-898e-ae1116bf3ff8).html |
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