An affinity-based approach to engineer laminin-presenting cell instructive microenvironments

Abstract

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices.

Introduction

In the framework of regenerative medicine and tissue engineering, much attention has been devoted towards the development of engineered matrices incorporating bioadhesive cues present in stem cell niches, to recapitulate the dynamic nature and biological complexity of these microenvironments, as well as gain more insight into the function of specific extracellular matrix (ECM) components on stem cell behavior. The ECM is an essential component of the stem cell niche, as it modulates important biological functions including proliferation, self-renewal and differentiation of stem cells. Among major ECM constituents, laminins play crucial and essential roles in many aspects of tissue physiology and function [[1], [2], [3], [4], [5]]. These heterotrimeric glycoproteins comprise several bioactive domains involved in the modulation of different biological functions. The latter include the interaction with other ECM proteins (e.g. nitrogen, netrin 4 and collagen VII) mediated by the laminin short arms (N-terminus), which contributes to the assembly and stability of basement membranes. These domains are also responsible for the laminin ability to polymerize [[6], [7], [8]], even in the absence of other basement membrane components, forming the molecular network that will be in contact with the cellular surface. In addition to its structural role, laminin comprises multiple bioactive domains that interact with cell surface receptors (e.g. integrins, dystroglycans, and syndecans), modulating different cell functions including cell adhesion, proliferation, migration and differentiation, as well as ECM deposition [[9], [10], [11]].

Laminin has been incorporated into both two-dimensional (2D) [12] and three-dimensional (3D) [[13], [14], [15], [16], [17], [18], [19], [20]] cell-instructive microenvironments for applications in regenerative therapies or to get insights into the role of laminin on the modulation of cell behavior. Strategies explored for laminin immobilization have relied either on its non-selective adsorption or entrapment or, alternatively, on its non-selective covalent immobilization to different substrates through the use of functional groups present in multiple sites of the laminin structure such as amines [[13], [14], [15], [16], [17]] and thiols [18,20]. One of the main caveats presented by these strategies is the inability to control orientation and conformation of laminin upon immobilization, which were proven to be crucial for the modulation of cellular behavior [[21], [22], [23]]. As such, the exposure of key laminin bioactive epitopes can be compromised. In an attempt to assure the control over the tethering of laminin, in recent years protein immobilization strategies have shifted toward site-specific conjugation, with special focus on biorthogonal chemical reactions (click chemistry), enzymatic ligation and affinity binding, using either unnatural amino acids or engineered site-selective amino acid sequences [[24], [25], [26]]. These strategies are expected to provide a higher retention of bioactivity, by favoring the access to the active sites of immobilized proteins. To the best of our knowledge, to date, only one study reported the site-selective immobilization of laminin [27]. To control the presentation of full-length ECM proteins without altering their bioactivity, Lee and co-workers explored click chemistry to anchor collagen, fibronectin and laminin onto polyacrylamide gels by their N-terminus [27]. Nevertheless, despite guaranteeing the site-selective immobilization of the proteins, as result of the use of laminin N-terminus, this approach compromises one of laminin's hallmark features, which is its ability to polymerize. Affinity-binding has been increasingly explored for the site-specific and reversible conjugation of proteins and peptides, because of its versatility and ability to generate dynamic biomimetic systems. However, the successful implementation of this strategy is strongly dependent on the appropriate selection of the binding pairs. Binding systems using high affinity interactions, such as streptavidin and biotin, although often used, require the protein of interest to be either recombinantly or chemically modified [28,29]. In contrast, the use of natural binding partners constitutes an attractive alternative, as strong non-covalent interactions can be achieved without the need for protein modification [30,31].

In this study, we examined an alternative strategy that explores the well described native high affinity interaction (K_D = 5 nM) between laminin and the N-terminal agrin (NtA) domain [32,33]. The agrin-binding site in laminin is localized in the central region of its coiled-coil domain and maps to a sequence of 20 conserved residues within the γ1 chain [33,34]. Interestingly, this interaction requires a coiled-coil conformation of the agrin-binding site [34]. To assess the ability of this affinity-based approach to immobilize laminin with retention of bioactivity, recombinant human NtA (rhNtA) domain was successfully produced and further conjugated, at its N-terminus, with a thiol-terminated poly (ethylene glycol) (PEG) to enable the preparation of self-assembled monolayers (SAMs) of NtA on gold. To the best of our knowledge, this is the first report on the production and characterization of the human variant of NtA domain. In order to study the specific interactions between the immobilized laminin and cells, SAMs were herein used as proof-of-concept platforms, since they can be easily produced and specifically tailored to provide a chemically well-defined molecular monolayer [35]. The binding ligand was characterized and its ability to mediate laminin immobilization through a high affinity interaction was shown by solid-binding assay, surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). The bioactivity of mono-PEGylated rhNtA-immobilized laminin was subsequently investigated by evaluating its ability to self-polymerize and modulate the behavior of human neural stem cells (hNSCs). hNSCs were herein used due to the well described role of laminin on the modulation of neural cell behavior [36]. This strategy represents a promising and versatile approach for the site-selective immobilization of laminin while preserving its bioactivity, which can be potentially useful for the development of cell instructive microenvironments for tissue engineering and regenerative medicine.