Construction of multifunctional fusion proteins with a laminin-derived short peptide to promote neural differentiation of mouse induced pluripotent stem cells.

ABSTRACT

There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage. The functionality of these chimeric proteins, referred to as ERE-p20 and E-p20, was assessed by determining their ability to immobilize cells on a hydrophobic polystyrene surface, improve mouse induced pluripotent stem cells (miPSCs) attachment, and promote miPSC differentiation to neural progenitors. ERE-p20 and E-p20 proteins showed hydrophobic binding saturation to the polystyrene plates around 500 nM (2.39 µg/cm2 ) and 750 nM (2.27 µg/cm2 ) protein concentrations, respectively. The apparent maximum cell binding to ERE-p20 and E-p20 was approximately 81% and 73%, respectively, relative to gelatin. For neural precursors, neurite outgrowth was enhanced by the presence of RGD and p20 peptides. The expression levels of neuronal marker protein MAP2 were upregulated approximately 2.5-fold and threefold by ERE-p20 and E-p20, respectively, relative to laminin. Overall, we have shown that elastin-mimetic fusion proteins consisting of p20 with and without RGD peptides are able to induce neuronal differentiation. In conclusion, our newly designed bioengineered fusion proteins allow preparation of specific bioactive matrices or coating/scaffold for miPSCs differentiation.