The present project aims at developing a new treatment for spinal cord injuries that affect the most caudal part of the spinal cord, the conus medullaris, and associated lumbosacral nerve roots, the cauda equina. Trauma to this part of the nervous system is associated with paralysis and impairments of bladder and bowel functions, in part due to loss of motor neurons. We have developed a model in the rat consisting of lumbosacral nerve root injuries that mimic the human condition. The injury consists of an avulsion of the ventral roots from the spinal cord surface. In this project, we grow human embryonic stem cells to develop into immature motor neurons, and these cells are transplanted into the spinal cord of rats with lumbosacral nerve root injuries and repair. To date, significant progress has been made. We have shown that 1) Large numbers of motor neurons can be grown in tissue culture and later be used for cell transplantation into the rat spinal cord; 2) Transplanted human motor neurons at different developmental stages show long-term survival of up to at least 10 weeks in the rat spinal cord ; 3) Transplanted human motor neurons are capable of extending axons into the repaired ventral roots as well as into intact ventral roots and thereby enter the peripheral nervous system; 4) Transplantation of human motor neurons in combination with surgical repair of injured ventral roots resulted in return of a step-like voiding pattern detected during voiding behavioral studies; 5) Functional testing of the bladder muscle did not show any cell transplant-associated adverse functions; 6) Sensory threshold testing did not detect any signs of mechanical allodynia or heat hyperalgesia. During the present three-month reporting period, we studied and analyzed bladder wall tissues in control and experimental groups to evaluate the end-organ for any possible adverse effects of cell treatment as well as for the possibility of any desired therapeutic effect. Our studies on bladder wall thickness and on the thickness of the bladder lining showed no differences between control groups and treatment groups that had received human motor neurons as cell transplants into the spinal cord. We conclude that the human cell-based therapies did not inflict any adverse effects on the bladder wall, which represents the end-organ in our injury and repair model. Continued and ongoing analysis will further characterize the survival and differentiation pattern of transplanted human motor neurons as well as the effects on functional and anatomical outcome measures.