Design and Adsorption of Modular Engineered Proteins to Prepare Customized, Neuron-Compatible Coatings

Neural prosthetic implants are currently being developed for the treatment and study of both peripheral and central nervous system disorders. Effective integration of these devices upon implantation is a critical hurdle to achieving function. As a result, much attention has been directed towards the development of biocompatible coatings that prolong their in vivo lifespan. In this work, we present a novel approach to fabricate such coatings, which specifically involves the use of surface-adsorbed, nanoscale-designed protein polymers to prepare reproducible, customized surfaces. A nanoscale modular design strategy was employed to synthesize six engineered, recombinant proteins intended to mimic aspects of the extracellular matrix proteins fibronectin, laminin, and elastin as well as the cell–cell adhesive protein neural cell adhesion molecule. Physical adsorption isotherms were experimentally determined for these engineered proteins, allowing for direct calculation of the available ligand density present on coated surfaces. As confirmation that ligand density in these engineered systems impacts neuronal cell behavior, we demonstrate that increasing the density of fibronectin-derived RGD ligands on coated surfaces while maintaining uniform protein surface coverage results in enhanced neurite extension of PC-12 cells. Therefore, this engineered protein adsorption approach allows for the facile preparation of tunable, quantifiable, and reproducible surfaces for in vitro studies of cell–ligand interactions and for potential application as coatings on neural implants

Cell-Binding Domain Context Affects Cell Behavior on Engineered Proteins

A family of artificial extracellular matrix proteins developed for application in small-diameter vascular grafts is used to examine the importance of cell-binding domain context on cell adhesion and spreading. The engineered protein sequences are derived from the naturally occurring extracellular matrix proteins elastin and fibronectin. While each engineered protein contains identical CS5 cell-binding domain sequences, the lysine residues that serve as cross-linking sites are either (i) within the elastin cassettes or (ii) confined to the ends of the protein. Endothelial cells adhere specifically to the CS5 sequence in both of these proteins, but cell adhesion and spreading are more robust on proteins in which the lysine residues are confined to the terminal regions of the chain. These results may be due to altered protein conformations that affect either the accessibility of the CS5 sequence or its affinity for the α_4β_1 integrin receptor on the endothelial cell surface. Amino acid choice outside the cell-binding domain can thus have a significant impact on the behavior of cells cultured on artificial extracellular matrix proteins

Lithographic Patterning of Photoreactive Cell-Adhesive Proteins

We describe a novel, simple method for the photolithographic patterning of cell-adhesive proteins. Intrinsically photoreactive proteins are synthesized in Escherichia coli through incorporation of the non-canonical, photosensitive amino acid para-azidophenylalanine. Upon ultraviolet irradiation at 365 nm, proteins form cross-linked films with elastic moduli that can be tuned by varying the concentration of photoreactive amino acid in the expression medium. Films of these proteins can be directly patterned using standard photolithographic techniques. We demonstrate the utility of this method of protein patterning by creating stable arrays of fibroblast cells on an engineered protein “photoresist”

Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-β signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer

Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture microenvironments

Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research

Bioprinting Cell- and Spheroid-Laden Protein-Engineered Hydrogels as Tissue-on-Chip Platforms

Human tissues, both in health and disease, are exquisitely organized into complex three-dimensional architectures that inform tissue function. In biomedical research, specifically in drug discovery and personalized medicine, novel human-based three-dimensional (3D) models are needed to provide information with higher predictive value compared to state-of-the-art two-dimensional (2D) preclinical models. However, current in vitro models remain inadequate to recapitulate the complex and heterogenous architectures that underlie biology. Therefore, it would be beneficial to develop novel models that could capture both the 3D heterogeneity of tissue (e.g., through 3D bioprinting) and integrate vascularization that is necessary for tissue viability (e.g., through culture in tissue-on-chips). In this proof-of-concept study, we use elastin-like protein (ELP) engineered hydrogels as bioinks for constructing such tissue models, which can be directly dispensed onto endothelialized on-chip platforms. We show that this bioprinting process is compatible with both single cell suspensions of neural progenitor cells (NPCs) and spheroid aggregates of breast cancer cells. After bioprinting, both cell types remain viable in incubation for up to 14 days. These results demonstrate a first step toward combining ELP engineered hydrogels with 3D bioprinting technologies and on-chip platforms comprising vascular-like channels for establishing functional tissue models

Matrix interactions modulate neurotrophin-mediated neurite outgrowth and pathfinding

Both matrix biochemistry and neurotrophic factors are known to modulate neurite outgrowth and pathfinding however, the interplay between these two factors is less studied. While previous work has shown that the biochemical identity of the matrix can alter the outgrowth of neurites in response to neurotrophins, the importance of the concentration of cell-adhesive ligands is unknown. Using engineered elastin-like protein matrices, we recently demonstrated a synergistic effect between matrix-bound cell-adhesive ligand density and soluble nerve growth factor treatment on neurite outgrowth from dorsal root ganglia. This synergism was mediated by Schwann cell-neurite contact through L1CAM. Cell-adhesive ligand density was also shown to alter the pathfinding behavior of dorsal root ganglion neurites in response to a gradient of nerve growth factor. While more cell-adhesive matrices promoted neurite outgrowth, less cell-adhesive matrices promoted more faithful neurite pathfinding. These studies emphasize the importance of considering both matrix biochemistry and neurotrophic factors when designing biomaterials for peripheral nerve regeneration

Spontaneous cardiomyocyte differentiation of mouse embryoid bodies regulated by hydrogel crosslink density.

Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration