Multivalent Effectors to Control Stem Cell Differentiation

Multivalent effectors to control stem cell differentiation

Numerous cellular signaling systems that regulate stem cell self-renewal and differentiation involve the assembly of multivalent ligands – oligomeric entities that present multiple binding sites and thereby bind multiple receptors on a target cell or stem cell – such as morphogens that naturally oligomerize, extracellular matrix protein engagement of integrins, and juxtacrine signaling between membrane-associated ligands and receptors. The resulting multivalent binding can be more potent than corresponding monovalent interactions, potentially through initiating a process of cellular receptor clustering that may promote enhanced signal transduction. However, the ability to control and monitor these naturally occurring multivalent interactions within a living cell is currently limited, since it is difficult to modulate the valency of naturally-occurring ligands, especially membrane-associated proteins. We have developed a means to synthetically conjugate the binding domains of normally cell membrane-associated ligands to the soluble biopolymer hyaluronic acid (HA). Specifically, using EDC/NHS chemistry, we functionalized the high molecular weight HA backbone and attached recombinantly produced ephrin-B ligand ectodomains via an added terminal cysteine. To precisely estimate the valency and molecular weight of the bioconjugates, we characterized the molecules using size exclusion chromatography and multi-angle light scattering.

To determine the effects of bioconjugate multivalency on the ability to cluster cell surface receptors, we incubated adult hippocampal neural progenitor cells (AHNPCs) with the multivalent molecule in vitro. Multivalent ephrin-B showed enhanced clustering of cognate Eph receptors as compared to antibody-clustered ligands. Next, to determine if increased receptor clustering could affect stem cell differentiation, cultures of AHNPCs were incubated solely with either antibody-clustered ligands or high conjugation ratio conjugates for six days. A maximum three-fold increase over the antibody-clustered ligand for the highest ratio conjugate was observed. To determine if the effect of multivalency in directing neuronal stem cell fate could be applied more generally to other cell types, cultures of human embryonic and induced pluripotent stem cells were incubated with the factors for 2-4 weeks. In both cultures a 2-fold increase in the fraction of neurons formed and a 4-fold increase in the fraction of dopaminergic neurons compared to antibody-clustered controls was observed. Cultures also showed increases in the midbrain-specific marker En1 and had significantly higher levels of dopamine. Finally, in an effort to validate the enhanced activity of this multivalent construct in vivo, we injected it directly into the brain of adult rats via intracranial stereotaxic injection. Brains injected with bioconjugates showed a three-fold increased ability to induce neuronal differentiation as compared to the antibody-clustered control, thus indicating that highly multivalent ligands potently enhance the fraction of new neurons formed compared to using ligands clustered by conventional means. These results have a variety of biomedical implications, in that they establish a general platform for creating highly bioactive, defined, and reproducible forms of protein-based ligands, which have the ability to more potently activate downstream effectors for use in basic research and therapeutic applications.