The potential of GDF5-functionalised collagen hydrogels to enhance dopaminergic cell replacement therapies for Parkinson's disease
Alamilla Marroquín, Verónica Rebeca
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Parkinson’s disease is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons from the substantia nigra and striatum. Over time, the gold standard pharmacotherapy, L-dopa, loses its efficacy in alleviating the motor symptoms that characterise this disorder, making it necessary to find alternative treatments that can modify the disease course or repair the damage already done. A promising reparative therapy for Parkinson’s disease is the transplantation of dopaminergic neurons either harvested from foetal tissue or derived from various stem cell sources. However, cell replacement therapies have been limited by poor survival leading to poor striatal reinnervation and limited motor recovery. Biomaterial-based therapies, like collagen hydrogels, offer significant potential to improve the outcome of cell replacement therapy by mimicking the in vivo microenvironment of the transplanted cells via giving them an adhesive matrix for growth and a supply of neurotrophic factors, while acting as a physical protection against inflammatory microglia and astrocytes. One potential neurotrophic factor to use in this context is growth differentiation factor 5 (GDF5) which is a potent dopaminergic neuroprotective agent. Therefore, the aim of this thesis was to determine if a dual approach combining the delivery of GDF5 and cell therapy in a collagen hydrogel could improve the outcome of cell replacement therapy in a rat model of Parkinson’s disease. Through a series of preliminary in vitro and ex vivo studies, we first assessed the biocompatibility of collagen hydrogels and the effects of GDF5 alone or GDF5-functionalised collagen hydrogels on a SH-SY5Y cell line and on a ventral mesencephalon (VM) explant system. Subsequently, we did preliminary assessments in vivo of the suitability of collagen hydrogels and GDF5-functionalised collagen hydrogels for the delivery of primary dopaminergic neurons (derived from the developing VM of the rat) at early time-points. Based on these results, we sought to investigate the long-term potential of GDF5-functionalised collagen hydrogels to enhance the survival, re-innervation capacity and function after transplantation of embryonic day 14 (E14) rat VM grafts in a rat model of Parkinson's disease. Additionally, we examined the long-term effects of GDF5+IL-10-functionalised collagen hydrogels to enhance the survival, re-innervation capacity and function after transplantation of E14 VM grafts in a rat model of Parkinson's disease. The in vitro and ex vivo results demonstrated that exposure to the collagen hydrogels did not have any detrimental effects on a human SH-SY5Y cell line or on explanted E14 rat VM tissue explants. Following this, we found that after a 6-hydroxydopamine (6-OHDA) insult on SH-SY5Y cells and VM explants, GDF5 treatment promoted recovery of cells. The preliminary in vivo studies demonstrated that collagen hydrogels are a suitable matrix for cell and neurotrophic factor delivery, that are well tolerated in the brain, and that support the survival of encapsulated primary cells. Subsequently, when we sought to determine the long-term survival and efficacy of E14 VM primary dopaminergic grafts in GDF5-loaded collagen hydrogels in a rat model of Parkinson's disease, we found that incorporation of GDF5, but not the collagen hydrogel, into the transplantation process actually reduced the survival, reinnervation and functional capacity of the grafted dopaminergic neurons. When we examined the long-term survival and efficacy of E14 VM primary dopaminergic grafts in GDF5+IL-10-loaded collagen hydrogels, we found that the addition of IL-10 did not have a detrimental effect on the grafts and we confirmed that the incorporation of GDF5 had damaging effects on the survival of the grafted dopaminergic neurons, suggesting that GDF5 was toxic to the transplanted cells. In conclusion, the results obtained in this thesis suggest that GDF5, at the dose used in this work, was detrimental to primary dopaminergic grafts. Nevertheless, collagen hydrogels functionalised with a different neurotrophic factor have the potential to enhance cell replacement therapies in Parkinson’s disease and should be further studied.