Development of three ELP constructs for cardiac stem cell therapy

Abstract

Introduction Myocardial infarction results in irreversible damage to heart muscle tissue, and current treatment strategies primarily focus on preventing secondary infarcts rather than regenerating the lost tissue. Consequently, a myocardial infarction often leads to cardiac arrhythmias and heart failure. New stem cell therapies, such as cardiac atrial appendage stem cells (CASCs), show promise [1]; however, their effectiveness is limited by poor cell retention, which prevents sufficient proliferation and differentiation into cardiomyocytes. 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) [2]. Recombinant 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 (RGD) is combined with a heparin binding domain (HBD). This peptide not only promotes focal adhesion formation, increasing interaction between scaffold and cells but also allows electrostatic interaction between the material and surrounding extracellular matrix.

Discussion Applying a Design of Experiments 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, IMAC revealed substantial nonspecific binding resulting in unsatisfactory purities for downstream applications. ITC and OEP, on the other hand, resulted in pure ELP samples as demonstrated via SDS-PAGE and SEC analyses. However, based on protein quantification using the BCA assay, ITC showed higher recovery yields, making it the method of choice. It should be noted, however, that OEP is far less time-consuming than three rounds of ITC.

Conclusions 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 lyate, ITC shows the best performance in comparison to IMAC and OEP in terms of yield and purity.

The purified ELPs’ are currently being characterised by e.g., measuring their transition temperatures through turbidity measurements. Preliminary biocompatibility are planned based on Human Umbilical Vein Endothelial Cells (HUVECs) in presence of ELP. Cell viability will be verified microscopically as well as via the Alamar blue assay.

Materials and Methods We have explored the biotechnological production of three ELP constructs in E. Coli. Optimization of the expression parameters (time, temperature, IPTG concentration) was done through a design of experiments (DoE) driven approach. After expression optimization, three different purification routes were compared: Immobilized metal affinity chromatography (IMAC), inverse transition cycling (ITC) and organic extraction and precipitation (OEP) [3]. After dialysis and freeze drying, molecular weight and purity were characterized through SDS-PAGE, Western Blot and MALDI-TOF.

Results Three tetrameric ELPs were expressed with constructs containing either only RGD (RRRR), only HBD (HHHH) or an equal mix of the two cell adhesion domains (RHHR). Expression optimization showed that standard protocols (37°C, 16h expression, 1 mM IPTG) in pLysS E. Coli are sufficient for RRRR and RHHR. HHHH however required a different approach. Following protein expression, IMAC, ITC (3 cycles) and OEP were compared regarding yield and purity through concentration determination and SDS-PAGE and SEC analyses.