There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo.

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling.