(122d) Multivalency Enhances the Potency of Recombinant Sonic Hedgehog in Dopaminergic Differentiation of Human Embryonic 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 dopaminergic and motor neurons from embryonic or induced pluripotent stem cells, which may be used in cell-based therapies for Parkinson's disease and spinal cord injury. Recent studies have demonstrated that endogenous Shh self-assembles to form potent, multimeric, and presumably multivalent molecules. Multivalent interactions have been shown to enhance cellular responses to extracellular stimuli, raising the possibility that multimerization modulates or enhances Shh activity. Unfortunately, commercial recombinant Shh produced in bacteria lacks the post-translational modifications necessary for multimerization. To circumvent this limitation, we have synthesized multivalent Shh molecules using recombinant Shh and HyA biopolymers, and have further demonstrated that multivalent HyA-Shh bioconjugates demonstrate enhanced potency on mammalian stem cells.

Using high molecular weight Hyaluronic acid (HyA) as a backbone, we conjugated recombinant Shh ligands at several valencies to synthesize multivalent HyA-Shh bioconjugates, thereby creating a versatile system to analyze the effects of ligand valency and spacing on potency. We have observed that HyA-Shh bioconjugates of particular valencies are 1-2 orders of magnitude more potent than their monovalent Shh subunit in inducing both differentiation of multipotent C3H/10T1/2 cells, an embryonic murine fibroblast cell line, and proliferation of adult rat neural stem cells. Of particular relevance to regenerative medicine applications, we have also demonstrated that optimal multivalent HyA-Shh bioconjugates can more efficiently instruct dopaminergic differentiation of human embryonic stem cells compared to monovalent or commercial recombinant Shh. Quantitative PCR analysis of side-by-side differentiation experiments revealed that human embryonic stem cells differentiated with HyA-Shh multivalent bioconjugates produce approximately 2-fold higher levels of mRNA encoding tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, than monovalent or commercial recombinant Shh. Furthermore, immunocytochemical analysis of differentiated cultures determined that HyA-Shh bioconjugate of 1:18.2 molar ratio could produce a significantly higher level of TH-positive neurons (54.7%±8.05) than both monovalent (14.9%±8.7) and commercial (26.7%±3.0) recombinant Shh. Utilization of multivalent HyA-Shh bioconjugates may thus further facilitate generation of dopaminergic and potentially motor neurons for regenerative medicine therapies for Parkinson's disease and spinal cord injury.