Transient spinal cord ischemia is a serious complication associated with aortic cross clamping (a surgical procedure required for the repair of aortic aneurysm). Neurological dysfunction resulting from transient spinal cord ischemia may be clinically expressed as paraparesis, fully-developed spastic paraplegia, or flaccid paraplegia. In spastic paraplegia, the underlying spinal pathology is characterized by a selective loss of inhibitory cells (neurons) in the ischemia-injured spinal cord. That loss of inhibition produces increased muscle tone (i.e. spasticity). While there are some current pharmacological treatments for spasticity that provide a certain degree of functional improvement, there are no effective therapies that lead to clinically-relevant, long-lasting recovery. One of the therapeutic approaches pursued by our group is the characterization of functional changes after spinal cord transplantation of neuronal cells previously generated in culture with the goal of replacing missing inhibitory neurons in the spinal cord. In our recent experiments, we characterized the survival and differentiation of human embryonic stem cell-derived neural precursors that were grafted into the spinal cord of rats with a previous spinal ischemic injury. Our initial data demonstrate that spinal grafting of neural precursors generated from 3 independent human embryonic stem cell lines is associated with long-term cell engraftment of grafted cells. A significant population of the grafted cells displayed neuronal differentiation, progressive maturation, and expression of markers which are typical for mature, functional human neurons. Initial analysis of grafted cells also indicated the development of functional connectivity between transplanted neurons and surviving neurons of the recipient. A significant advancement in our effort to characterize the effect of such a treatment was the use of a sorting technique which permits the generation of large quantities of highly-purified neural precursors. The capacity to generate such large quantities of pure cell populations is particularly important in our large preclinical animal model (minipig), which is essential to move this therapeutic approach to clinic. In addition, we characterized an efficient cell freezing protocol. The sorting and freezing techniques together allow large quantities of identical cell populations to be frozen for future transplantation, ensuring a group of animals receives an identical cell population. Our plan for the next year is to perform long-term functional recovery studies in our minipig model of spinal ischemia.