ENZYME TRIGGERED SELF-ASSEMBLED PEPTIDE DERIVATIVE HYDROGELS FOR EMBRYONIC STEM CELL CULTURE

Aromatic peptide amphiphiles that self-assemble in response to a trigger, such as pH or enzymes, have the ability to support the culture of somatic cell types, in both two-dimensional (2D) and three-dimensional (3D) culture. Although a fully defined synthetic substrata is required for the successful clinical applications of Embryonic Stem (ES) cells hydrogels of SA aromatic peptide amphiphiles have not been investigated for this purpose. The aim of this investigation is to produce alkaline phosphatase (AP) triggered hydrogels as a substratum for ES cell culture. This SA trigger was chosen as it utilizes inherent biological processes, through the enhanced AP activity of ES cells, with SA occurring in otherwise constant conditions. We also sought to overcome the current inability to consistently control ES cell behaviour in vitro through two different routes that have previously been demonstrated to effect stem cell culture. Firstly, control of the hydrogels mechanical properties and secondly through the incorporation of biological function, principally through the addition of glycosaminoglycans (GAGs). Firstly AP triggered hydrogels of 9-fluorenylmethoxycarbonyl-Tyrosine-OH (Fmoc-Y-OH) were studied and compared with those formed by pH trigger. An unexpected relationship between AP concentration and molecular order was detected. It was observed that the hydrogels stiffness was controlled through the AP concentration; ideal for ES cell culture as Engler et al. (2006) has previously demonstrated the effect material stiffness had on the differentiation pathways chosen by mesenchymal stem cells. Differences between the SA trigger were detected with the hydrogels formed by pH trigger exhibiting significantly lower mechanical properties. This was attributed to the SA process and the disorder that arises from forming all of the hydrogelators instantaneously. The SA process of AP triggered Fmoc-Y-OH showed a 4 stage process. The first stage, dephosphorylation, occurred in a time and AP concentration dependent manner. The second stage transpired due to the spontaneous SA of Fmoc-Y-OH providing a temporary change in fluorenyl environment. The final stages, formation of chiral one dimensional (1D) fibres through proposed ? -? interactions and gelation through the entanglement of fibres are closely linked.Secondly we investigated AP triggered Fmoc-Phenylalanine-Tyrosine-OH (Fmoc-FY-OH) hydrogels in two distinct physiological environments. When formed in buffer (0.15M, pH 7) an optimum AP concentration was observed in terms of molecular interactions, ? -? interactions, which translated to the hydrogels mechanical properties. In these conditions helical fibres were imaged by AFM. The second medium for SA was KnockOUT? DMEM, developed for ES cell culture. Although similarities in the molecular interactions were detected it appears the SA environment effects the structures formed with non-helical fibres imaged. GAGs were successfully incorporated into the hydrogels at biologically relevant levels at the extremes of sulphation. The sulphation patterns of the GAGs secreted by ES cells changes during differentiation and may provide a way to guide cell behaviour through growth factor binding. However the GAGs were not entrapped in the fibre network and leached out into solution limiting their ability to guide ES cell behaviour. Unfortunately both of the hydrogels produced in this study were deemed unsuitable as ES cell substrata due to their instability (Fmoc-Y-OH) or low biocompatibility (Fmoc-FY-OH). However we have demonstrated that it was possible for endogenous AP to trigger SA, indicating that in the future ES cells may be able to form their own substrata preventing the need for exogenous AP.