Multivalent ligands control stem cell behaviour <i>in vitro</i> and <i>in vivo</i>

There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions1–7. 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 biology8–10. 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 clustering11–18. 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. Adult neural stem cells (NSCs) are an important class of therapeutically relevant cells that persist in specific regions of the mammalian brain, and have the capacity to generate new neurons and glia throughout life19. Human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the capacity to differentiate into all cells of the adult body and therefore offer broad potential for cell replacement therapy and modelling of human disease. We recently found that ephrin-Eph signalling regulates both the neuronal differentiation of adult hippocampal NSCs19 and the differentiation of hESCs into dopaminergic neurons20, cells lost in Parkinson’s disease. The design of molecules that regulate ephrinEph signalling in NSCs and hPSCs could therefore advance both basic biology and therapeutic applications. To create synthetic multivalent ligands with potentially high potencies, recombinantly produced ephrin-B2 extracellular domain was conjugated at a range of stoichiometries to highmolecular-weight hyaluronic acid (HA)—a well-characterized biopolymer present throughout the body and in particular within the brain—using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/sulfo-NHS chemistry, as described previously21 (Fig. 1a,b). The valencies of the resulting 100 nm polymeric conjugates22—estimated using a bicinchoninic acid (BCA) assay and further quantified with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS; Fig. 1c)— ranged from 2 to 25 ephrin molecules per HA chain in this synthesis. Based on the recently discovered role of ephrin-B2 signalling in regulating neuronal lineage commitment of adult NSCs19, we investigated the activity of the multivalent conjugates in NSC culture. At a given concentration of ephrin-B2 ectodomain molecules, increasing the valency of HA:ephrin-B2 conjugates progressively elevated neuronal differentiation (Fig. 1d,e). Strikingly, compared to the antibody-clustered ligand, the 1:22, 1:12 and 1:8 valency conjugates induced similar levels of neuronal differentiation at 37-, 26and 9-fold lower protein concentrations, respectively. Thus, in contrast to the current standard antibody-clustered form, for which the low potency necessitates high concentrations, the multivalent ligands are potent agonists, thereby potentially reducing costs. Next, the addition of monomeric ephrin-B2 in tenfold molar excess to Fc-ephrin-B2 wells blocked differentiation, further establishing that ephrin clustering is required for activity. Finally, the results were further validated by quantifying mRNA levels of the neuronal marker Tubb3 (Fig. 1f ). We next compared the ability of natural and synthetic ligands to cluster Eph receptors. Because ephrin-B2 presented from astrocytes regulates the neuronal differentiation of adult NSCs19, we analysed ephrin-Eph localization on NSCs in contact with hippocampal astrocytes. Punctate staining of both ephrin-B2 and its receptor EphB4 was observed at cell–cell junctions (Fig. 2a), and co-localization of the ligand and receptor was also observed at cell–cell contacts in the subgranular zone (SGZ) of the adult hippocampus (Fig. 2b), where NSCs reside19. We analysed whether the multivalent conjugates could emulate this natural process of receptor–ligand assembly. Fluorescently labelled ephrin-B2 conjugates were synthesized and incubated with NSCs, at 4 8C to block endocytosis. EphB4 localization was diffuse across the cell membrane in the absence of ephrin-B2 or with low-ratio conjugates, whereas EphB4 puncta were observed in the presence of highly multivalent conjugates or antibody-clustered ligand (Fig. 2c). Additionally, although low ephrin-B2 valency conjugates yielded fewer and smaller EphB4 clusters than