Mia L. Huang

Glycocalyx Remodeling with Proteoglycan Mimetics Promotes Neural Specification in Embryonic Stem Cells

Growth factor (GF) signaling is a key determinant of stem cell fate. Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs). Here, we report a cell surface engineering strategy that exploits the function of HSPGs to promote differentiation in embryonic stem cells (ESCs). We have generated synthetic neoproteoglycans (neoPGs) with affinity for the fibroblast growth factor 2 (FGF2) and introduced them into plasma membranes of ESCs deficient in HS biosynthesis. There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification. This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

Priming the Cellular Glycocalyx for Neural Development

Glycans are important contributors to the development and function of the nervous system with enormous potential as therapeutic targets. However, a general lack of tools for tailoring the presentation of specific glycan structures on the surfaces of cells has left them largely unexplored in the biomedical context. In this Viewpoint, we briefly summarize the distinct challenges and complexities of the Glycome. We also highlight an emerging concept of cell surface engineering using synthetic nanoscale mimetics of native glycoconjugates to harness some of the unique biology of glycans, with an eye toward advancing stem cell-based neuroregenerative therapies.

Glycomaterials for probing host–pathogen interactions and the immune response:

The initial engagement of host cells by pathogens is often mediated by glycan structures presented on the cell surface. Various components of the glycocalyx can be targeted by pathogens for adhesion to facilitate infection. Glycans also play integral roles in the modulation of the host immune response to infection. Therefore, understanding the parameters that define glycan interactions with both pathogens and the various components of the host immune system can aid in the development of strategies to prevent, interrupt, or manage infection. Glycomaterials provide a unique and powerful tool with which to interrogate the compositional and functional complexity of the glycocalyx. The objective of this review is to highlight some key contributions from this area of research in deciphering the mechanisms of pathogenesis and the associated host response.

Synthetic Mucus Nanobarriers for Identification of Glycan-Dependent Primary Influenza A Infection Inhibitors

Current drugs against the influenza A virus (IAV) act by inhibiting viral neuraminidase (NA) enzymes responsible for the release of budding virions from sialoglycans on infected cells. Here, we describe an approach focused on a search for inhibitors that reinforce the protective functions of mucosal barriers that trap viruses en route to the target cells. We have generated mimetics of sialo-glycoproteins that insert into the viral envelope to provide a well-defined mucus-like environment encapsulating the virus. By introducing this barrier, which the virus must breach using its NA enzymes to infect a host cell, into a screening platform, we have been able to identify compounds that provide significant protection against IAV infection. This approach may facilitate the discovery of potent new IAV prophylactics among compounds with NA activities too weak to emerge from traditional drug screens.

Glycocalyx Remodeling with Glycopolymer-Based Proteoglycan Mimetics.

The cellular glycocalyx controls many of the crucial signaling pathways involved in cellular development. Synthetic materials that can mimic the multivalency and three-dimensional architecture of native glycans serve as important tools for deciphering and exploiting the roles of these glycans. Here we describe a chemical approach for the engineering of growth-factor interactions at the surfaces of stem cells using synthetic glycomimetic materials, with an eye towards promoting their commitment towards specific cell lineages with therapeutic potential.

Small Molecule Antagonist of Cell Surface Glycosaminoglycans Restricts Mouse Embryonic Stem Cells in a Pluripotent State

Recently, the field of stem cell‐based regeneration has turned its attention toward chemical approaches for controlling the pluripotency and differentiation of embryonic stem cells (ESCs) using drug‐like small molecule modulators. Growth factor receptors or their associated downstream kinases that regulate intracellular signaling pathways during differentiation are typically the targets for these molecules. The glycocalyx, which plays an essential role in actuating responses to growth factors at the cellular boundary, offers an underexplored opportunity for intervention using small molecules to influence differentiation. Here, we show that surfen, an antagonist of cell‐surface glycosaminoglycans required for growth factor association with cognate receptors, acts as a potent and general inhibitor of differentiation and promoter of pluripotency in mouse ESCs. This finding shows that drugging the stem cell Glycome with small molecules to silence differentiation cues can provide a powerful new alternative to existing techniques for controlling stem cell fate. Stem Cells 2018;36:45–54

Glycocalyx Scaffolding to Control Cell Surface Glycan Displays

This article describes a protocol for remodeling cells with synthetic glycoprotein and glycolipid mimetics that are functionalized with lipid anchors, allowing for cell surface display of specific glycan structures in predefined nanoscale arrangements. The complex chemical heterogeneity of glycans found on the cell surface or the glycocalyx renders analysis of the individual contributions of glycans difficult. This technique allows for the precise study of individual glycans at different regions of the glycocalyx, and may be useful for interrogating glycan interactions in infection or immunity or in stem cell differentiation. CHO‐Lec2 cells are prepared as adherent monolayers and, after reaching confluence, are incubated with the glycomaterials. Synthetic glycopolymers bearing α‐2,3‐sialyllactose glycans are used to decorate cellular surfaces in the form of 3D multivalent ligands projecting away from the cell surface, while α‐2,6‐sialyllactose glycolipid conjugates are used to anchor glycans in dynamic 2D arrays proximal to the cell membrane. Following washing, mimetic incorporation and glycan display can be analyzed using lectins with specificity for α‐2,3‐ or α‐2,6‐linked sialic acids. Flow cytometry data reveals that cell surface remodeling with either glycoconjugate mimetic occurs efficiently in a dose‐dependent manner. Combinations of glycoconjugates can also be employed simultaneously to generate a mixed glycocalyx with tunable composition and organization.

Influencing early stages of neuromuscular junction formation through glycocalyx engineering

Achieving molecular control over the formation of synaptic contacts in the nervous system can provide important insights into their regulation and can offer means for creating well-defined in vitro systems to evaluate modes of therapeutic intervention. Agrin-induced clustering of acetylcholine receptors (AChRs) at postsynaptic sites is a hallmark of the formation of the neuromuscular junction, a synapse between motoneurons and muscle cells. In addition to the cognate agrin receptor LRP4 (low-density lipoprotein receptor related protein-4), muscle cell heparan sulfate (HS) glycosaminoglycans (GAGs) have also been proposed to contribute to AChR clustering by acting as agrin co-receptors. Here, we provide direct evidence for the role of HS GAGs in agrin recruitment to the surface of myotubes, as well as their functional contributions toward AChR clustering. We also demonstrate that engineering of the myotube glycocalyx using synthetic HS GAG polymers can replace native HS structures to gain control over agrin-mediated AChR clustering.

Embryonic stem cell engineering with glycomimetic FGF2/BMP4 co-receptor drives mesodermal differentiation in 3D culture

Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.

Heparin-fibronectin interactions in the development of extracellular matrix insolubility

During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FNs multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FNs major heparin-binding domain HepII (modules III12-14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.