Peptide Hydrogels for Advanced 3D Cell Culture

Abstract

Stem cell technology is an invaluable tool in tissue engineering and regenerative medicine, with induced pluripotent stem cells (iPSCs) providing the means to design: autologous cell therapies, drug screening platforms and disease/ developmental models. However, the expansion of hiPSCs in vitro typically relies on either the presence of a layer of supportive feeder cells, or the use of ill-defined matrices. Moreover, following differentiation, iPSC-derivatives often lack the same phenotypic traits as their native counterparts. As such, the field of tissue engineering moves toward recapitulating the cell niche in vitro, through the design of biomaterials for 3D cell culture. With this in mind, the designs of new biomaterials are emerging all the time. Of particular focus are 'hydrogels'; a water-swollen network of polymeric fibres which entangle and/ or aggregate to form a self-supporting construct. These fibres can be made of natural and/ or synthetic polymers. Of great interest are peptide hydrogels. Peptide sequences can be designed to self-assemble into an array of larger macromolecular structures. One family of self-assembling peptide hydrogels (SAPHs) are based on an alternating sequence of hydrophilic/ hydrophobic amino acid residues which self-assemble to form beta-sheet nanofibres. Many variations of these SAPHs currently exist within the literature, e.g. EAK, RADA and KFE, and have already shown great promise in supporting the culture of many different cell lineages; a promising substrate for stem cell culture and tissue engineering applications. Importantly, when considering any biomaterial for 3D cell culture, it is vital to determine whether the abundance of material interferes with the method of analysis, either through interactions with the substrate of interest, or a component(s) of the technique. The work presented here is the first to outline the development of protocols for the following applications: 1) the extraction of RNA from cells encapsulated in SAPHs for downstream quantitative polymerase chain reaction (qPCR); and 2) the solubilisation of SAPHs and cell proteins for downstream western blot analysis. For RNA extraction, RNA was isolated from four peptide formulations - which vary in net charge - using either: 1) precipitation-based extraction, or 2) solid-state RNA binding to a silica membrane. The latter was more effective at removing contaminants but both methods resulted in a low yield - demonstrating a clear interference from the presence of SAP. However, enzymatic degradation of the hydrogel construct prior to RNA isolation significantly increased the amount of RNA recovered. On the other hand, for western blot analysis, the focus was on achieving complete solubilisation of both the self-assembling peptide (SAP) monomer and cell proteins following disruption of the hydrogel construct and the lysis of encapsulated cells. The use of urea - unlike a detergent-based buffer (Radioimmunoprecipitation assay buffer, aka RIPA) - in combination with multiple cycles of ultrasonication, was able to completely solubilise the SAP; thereby enabling downstream western blot analysis of cell proteins which are otherwise lost to SAP aggregation. The presence of SAP did not interfere with the standard immunoblotting technique. Together, these methods enable accurate determination of changes in gene/ protein expression of cells encapsulated in SAPH to better understand how cell-matrix interactions influence cell behaviour. The modified protocols were then used to determine the suitability of SAPHs for 3D stem cell expansion and iPSC-to-cardiomyocyte differentiation. The cells expressed pluripotency markers following encapsulation and short-term 3D culture. For cardiomyocyte differentiation, the protocol requires recreating the biphasic Wnt signalling which drives cardiomyogensis in vivo. In this study, this was achieved through the addition of a Wnt activator (Chir99021) and subsequent addition of a Wnt inhibitor (Wn

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alberto Saiani (Supervisor) & Delvac Oceandy (Supervisor)