469343 Biomaterials for Human Pluripotent Stem Cell Derived Midbrain Dopaminergic Neuron Generation and Transplantation to Treat Parkinson’s Disease
Human pluripotent stem cells (hPSCs) were cultured and differentiated into mDA neurons in 3D pNIPAAm-PEG gels. The authenticity of resulting neurons thoroughly characterized by immunocytochemistry, qPCR and voltage-sensitive dye based electrophysiology, and benchmarked against mDA neuronal development on 2D surfaces. Subsequently, a biodegradable, hyaluronic acid (HA) based hydrogel functionalized with ECM derived factors was developed for maturation and transplantation of mDA neurons generated on pNIPAAm-PEG. Post-transplantation survival of hydrogel-encapsulated neurons was then investigated 4.5 months after striatal implantation into rats.
mDA neuron production typically involves undefined components and difficult to scale-up 2D culture formats. Here, we used a fully-defined, 3D thermoresponsive biomaterial platform to derive mDA neurons from PSCs at substantially greater yield, quality, and maturity compared to 2D Matrigel coated surfaces. In particular, after 25 days of differentiation, ~40% of cells differentiated in 3D expressed tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine production and a hallmark of mDA neurons, compared to 20% on 2D. Crucial for functional mDA neurons, ~ 5-fold higher proportion of neurons differentiated in 3D fired mDA-like action potentials. Interestingly, FOXA2 and EN1, markers known to improve survival of mDA neurons, were expressed at significantly higher levels in cells generated in the 3D platform compared to control 2D surfaces.
Low post-transplantation cell survival is often attributed to adverse biochemical, mechanical, and immunological stresses cells experience during and post-transplantation. To address these challenges, we developed a functionalized hyaluronic acid (HA) based hydrogel first to mature pluripotent stem cell (PSC) derived neural progenitors towards functional neurons in vitro in a biomimetic 3D environment, and second to effectively transplant hydrogel-encapsulated neurons into the central nervous system. We demonstrated that incorporating adhesive peptides to promote cell adhesion increased extended neurite density, and adding neurotrophic factors increased dopaminergic differentiation in our 3D HA gels. Furthermore, we observed higher survival post-harvest and injection in vitro for mDA neurons matured within hydrogels compared to those on 2D surfaces. By using an optimized 3D HA biomaterial matrix for neuronal maturation and transplantation, we significantly increased the post-transplantation survival of hPSC derived mDA neurons in the rat striatum by 5 fold compared to the current standard method of bolus injections of unencapsulated neurons.
The combination of a defined, scalable, and resource-efficient cell culture platform and an optimally engineered transplantation biomaterial significantly increased the yield of mDA neurons generated in vitro and improved their post-transplantation viability in vivo, thereby addressing crucial challenges in treatment of neurodegenerative disease.