Development of elastin-like protein constructs for cardiac stem cell therapy

Abstract

Myocardial infarction results in irreversible damage to heart muscle tissue, and current treatment strategies primarily focus on preventing further complications rather than regenerating the lost tissue. New stem cell therapies, such as cardiac atrial appendage stem cells (CASCs), show promise; however, their effectiveness is limited by poor cell retention. Injectable hydrogels offer an elegant approach to enhance stem cell retention. Dynamic hydrogel networks can be formed by combining elastin-like protein (ELP) with hyaluronic acid (HA). ELP offers excellent tunability, allowing control over the gel’s mechanical properties and providing LCST behavior, which causes the gel to stiffen at body temperature. Functionalizing ELP with hydrazide groups allows crosslinking with aldehyde-functionalized HA, forming hydrazone bonds that confer the hydrogel's injectability. Due to the recombinant character of ELP, it is possible to incorporate multiple bioactive peptide sequences into the construct, allowing for synergistic effects in terms of biological activity. In this work, the universal cell attachment peptide sequence arginine-glycine-aspartic acid (R) is combined with a heparin-binding domain (H). We have explored the biotechnological production of three ELP constructs in E. Coli. Optimization of the expression parameters was done through a design of experiments (DoE) driven approach. Next, three purification routes were compared: Immobilized metal affinity chromatography (IMAC), inverse transition cycling (ITC) and organic extraction and precipitation (OEP). Molecular weight and purity were characterized through SDS-PAGE, Western Blot and ESI-MS. Human Umbilical Vein Endothelial Cells (HUVECs)are grown in the presence of ELP to verify cell viability via the Alamar blue assay. Applying a DoE approach allowed us to efficiently define the ideal expression conditions for our ELP constructs. With respect to the recovery of the ELPs from the cell lysate, OEP revealed substantial ELP loss, resulting in unsatisfactory yield for downstream applications. ITC and IMAC, on the other hand, resulted in pure ELP samples of sufficient yield. Three tetrameric ELP constructs were successfully expressed in E. Coli and optimal expression parameters were derived using a Design of Experiments approach. Considering the recovery of ELPs from the cell lysate, ITC shows the best performance in comparison to IMAC and OEP in terms of yield and purity. The purified ELPs are currently being characterized by e.g., measuring their transition temperatures through turbidity measurements, DSC and impedance-based sensing. Additionally, cell studies are being performed to assess the effect of the bioactive domains on viability and proliferation.