(117e) Multivalency Enhances the in Vitro Potency of Recombinant Sonic Hedgehog On Stem Cells

In vivo, Sonic hedgehog (Shh) plays a critical role as a morphogen in patterning neuronal differentiation in the developing neural tube, midbrain, and forebrain and as a mitogen in regulating the proliferation of neural stem cells in the adult nervous system. Due to its critical roles in neural development, it is ubiquitously employed in ex vivo systems to derive motor and dopaminergic neurons from embryonic or induced pluripotent stem cells, which have promise for therapies to treat Alzheimer's and Parkinson's disease patients, respectively. Recent in vitro studies have demonstrated that natural, endogenous Shh self-assembles to form potent, multimeric, and presumably multivalent molecules. Multivalent molecules can simultaneously bind multiple receptors on a cell, and multivalent interactions have been shown to enhance cellular responses to extracellular stimuli, raising the possibility that multimerization modulates or enhances Shh activity. Unfortunately, recombinant Shh produced in bacteria lacks the post-translational modifications necessary for self-assembly. Using high molecular weight Hyaluronic acid (HyA) as a backbone, we conjugated recombinant Shh ligands at several valencies to create multivalent HyA-Shh bioconjugates, thereby creating a versatile system to analyze the effects of ligand valency and spacing on potency. We have demonstrated that HyA-Shh bioconjugates are ~1-2 orders of magnitude more potent than monovalent Shh in inducing both differentiation of multipotent C3H/10T1/2 cells, an embryonic murine fibroblast cell line, and proliferation of adult neural stem cells. Furthermore, it appears the greatest increase in potency occurs at an optimal, mid-level Shh valency, possibly suggesting a nanoscale organization of the Shh receptor, Patched, within the cell membrane. Lastly, we will discuss the results of ongoing studies comparing the efficacy of monovalent recombinant Shh and multivalent HyA-Shh bioconjugates in instructing dopaminergic differentiation of human embryonic stem cells. Utilization of multivalent Shh bioconjugates may improve stem cell-based therapies for Parkinson's and Alzheimer's disease patients, and may be used as a tool to further basic scientific understanding of the Sonic hedgehog signaling pathway.