CELL TRANSPLANTATION · Use of embryonic stem cells and organotypic spinal cord slice preparations to establish cell-based therapies for spinal cord injury

Final Activity Report Summary - CELL TRANSPLANTATION (Use of embryonic stem cells and organotypic spinal cord slice preparations to establish cell-based therapies for spinal cord injury)

Cell transplantation has become a promising treatment for neurodegenerative diseases and trauma in the Central nervous system (CNS). The overall objective of this project was to investigate and optimise possible therapeutic strategies for Spinal cord injury (SCI) via combination of cell-based therapies with concurrent or sequential application of neuroprotective and proregenerative factors. Animal models for SCI were valuable for the advance in understanding cellular mechanisms that promoted neuroprotection and regeneration. However, due to the complexity of in vivo systems in providing easy result interpretations and the increased cost-effectiveness, in vitro systems might be advantageous for initial assays. For this purpose, we generated two neurotrauma models of SCI by using organotypic cultures of spinal cord, which maintained the basic cord cytoarchitecture and dorsal-ventral orientation. Considering that excitatory aminoacids were proposed as a potential cause for secondary injury after SCI, one injury model was based on the induction of neuronal death by the application of 50 µM glutamate. The second model of in vitro neurotrauma was based on the application of a mechanical insult by rolling a cylinder on the slice. Immunohistochemical analysis confirmed that these models caused 96.1 % and 95.8 % of neuronal death, respectively. These models were used to analyse the neuroprotective effect of FK506, a commonly used immunosuppressant drug. We found that the general cytoarchitecture of the spinal cord explants was better preserved in the presence of FK506 after excytotoxic induction, however only mild neuronal survival was obtained, equal to 8.9 %. Considering that the number of neurons recovered was still modest, we attempted to implement a combined strategy via the use of cell grafting to restitute lost cells. Thus, we employed neural-derived embryonic stem cells for this purpose. Before the grafting experiments we tried to improve a classical protocol for neural Embryonic stem (ES) cell differentiation in order to enrich the production of cholinergic neurons. Cholinergic neurons could potentially substitute motoneurons that were lost in the injured spinal cord. We succeeded in promoting the observation that all neurons obtained by ES cells expressed a cholinergic phenotype, namely Choline acetyl transferase (ChAT) plus, by using the morphogen sonic hedgehog and retinoic acid. Then, pre-differentiated ES cells were placed on control and injured spinal cord slices to assess for their differentiation after grafting. These cells were also grafted on the neurotrauma in vitro models in combination with FK506. The analyses of these assays were in progress by the time of the project completion. We also assessed the neuroprotective potential of different drugs. The results obtained from these studies were anticipated to provide valuable information about the action of different drugs on neuronal survival and would allow us to establish the optimal therapeutic combination to subsequently explore in vivo animal models for SCI.

Data: CORDIS, © European Union