The PI proposes an innovative approach to generate enriched populations of dopaminergic (DA) neurons for cell therapies. Idiopathic death of DA neurons causes the symptoms of Parkinson's disease (PD), thus, replacement of these cells is a primary goal of PD research. For cell replacement therapies, currently fetal derived cells show the most promising results in experimental models as well as in clinical trials. However, in clinical studies some patients developed graft induced dyskinesia (GID). These negative behavioral outcomes are thought to be due in part to the heterogeneity of the implanted cells. Therefore, developing protocols to obtain a more uniform population of NPCs for future transplantation studies will be extremely valuable. Here, a method is proposed for separation of DA precursors from ventral mesencephalic neural progenitor cells (VM NPCs) prior to ex vivo expansion and differentiation in 3D, hyaluronic acid (HA) hydrogels, which serve as biomimetic culture environments and possibly as cell transplantation vehicles.
Specific Aim 1 will exploit the specific interactions between the cell surface receptor CD44 and the glycosaminoglycan HA. Both HA and CD44 are overexpressed in fetal brain and down regulated during development. Preliminary results in the PI's lab demonstrate that HA-coated surfaces seeded with mixed cultures selectivity bind immature NPCs. HA-coated surfaces of varying concentrations will be used to pan for neuronal precursors from primary isolations of VM NPCs. Adhered and non-adhered cells will be separated and analyzed for DA precursor (Nurr1+), proliferation and differentiation potentials using immunostaining and RT-PCR. In parallel, CD44 expression of NPCs will be analyzed using flow cytometry and threshold values of CD44 expression for different NPC populations will be established.
In Specific Aim 2, pre-separated NPCs enriched in DA precursors will be cultured in 3D, HA hydrogels previously developed by the PI to enhance differentiation of VM NPCs into neurons. These hydrogels have been designed to provide a biomimetic environment in which the mechanical and chemical properties closely resemble those of native fetal brain. Additionally, native ECM proteins (e.g., laminin, fibronectin) will be added into the hydrogels for better cell adhesion and differentiation. Immunostaining and RT-PCR will be used to characterize differentiation of DA precursors in 3D cultures. The innovation of this project lies in the combination of pre-selection for DA precursors prior to ex vivo expansion and subsequent differentiation in 3D, HA biomaterials. The PI hypothesizes that large numbers of DA neurons, sufficient for therapeutic benefit, can be generated using this two-step approach.
The goal of this project is to develop strategies to expand, differentiate and purify neural progenitor cells (NPCs) in sufficient quantities for use in regenerative medicine. For example, this could be useful ultimately for therapies to treat central nervous system (CNS) disorders such as Parkinson's disease. In general, efficient expansion and functional differentiation of NPCs ex vivo poses a significant challenge. The first objective of this proposal is to enhance the purity of NPC cultures by panning primary isolates using selective hyaluronic acid matrix interactions. The second objective is to enhance the efficiency of neuronal differentiation from these purified NPC populations by providing appropriate matrix and soluble cues. The development of technologies to purify primary isolated stem cells and to subsequently control their differentiation potential in culture would be a vast opportunity for advancements in stem cell biology.