Introduction: Mesenchymal stem cells (MSCs) have been studied and used for cell-based therapy to regenerate heart tissues. However, MSCs cultured on tissue culture polystyrene (TCPS) shows low myocardial differentiation efficiency. Recent studies have reported that MSCs cultured in three dimensional (3D) conditions (e.g., in synthetic polymer hydrogels) express higher levels of myocardial marker genes than the cells cultured on TCPS. These results suggest that the 3D condition is promising for the myocardial differentiation of MSCs. However, low studies have worked on gels composed of proteins, which can be functionalize easily, as scaffolds to induce the differentiation. Here, we prepared three types of recombinantly-engineered elastin-like proteins (ELPs) (E-1, E-2, and E-3) mainly composed of repeated VPGIG. E-1, E-2, and E-3 proteins contain MMP2-cleavage peptides, cell-adhesive RGD peptides, and TGF-β1-binding peptides and MMP2-cleavage peptides, respectively. By using a cross-linker, each ELP was gelled, where rat MSCs were cultured to induce the myocardial differentiation.
Materials and Methods: ELPs were produced in transgenic E. coli and purified with a Ni chelate column. E-1, E-2, and E-3 gels were formed by adding a cross-linker, BS(PEG)9 (Pierce) at protein/BS(PEG)9 (wt%) of 7.50/0.92, 5.65/0.23, and 2.60/0.14, respectively, to make their Young’s moduli ~100 Pa (Young’s modulus of embryonic heart). Rat MSCs were cultured on TCPS and in ELP gels. At day 3, 5-azacytidine was added to the medium at the final concentration of 10 μM to induce the myocardial differentiation. At days 1 and 7, live and dead MSCs were stained with Calcein AM and Ethidium Homodimer III, respectively. At days 7 and 14, the gene expression level of MEF2D (myocyte-specific gene) was quantified by real-time PCR.
Results: Live/dead staining showed that almost all the MSCs were alive at day 1, and most MSCs were still alive at day 7. E-1 gels with MSCs collapsed clearly between days 7 and 14. The MEF2D gene expression level of ELP gel groups tended to be higher than that of TCPS groups at days 7 and 14. Especially, E-2 group showed significantly higher level than the TCPS groups.
Discussion: Gel formation procedure and culture in the gel had little effects on MSCs’ survival. TGF-β1-binding peptides in ELPs showed no significant effects, but MMP2-cleavage peptides may lead to the replacement of ELPs with matrices produced by MSCs, and RGD peptides was expected to promote the cardial differentiation of MSCs. Integrin α5β1-mediated signaling, which is reported to stimulate early cardiac development, might be activated by the RGD in ELPs.
Conclusion: ELP gels with RGD peptides could enhance the myocardial differentiation of MSCs.