Repair of Conus Medullaris/Cauda Equina Injury using Human ES Cell-Derived Motor Neurons

Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries. In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases. For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonoic During the first year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assess after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recorings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been submitted to a scientific journal and reviewed, and the manuscript is currently undergoing our revisions before being considered for publication. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also begun experiments using our refined model and cells, which now can be produced in high numbers and be identifiable during both the cell culture steps and during the animal studies. Initial tissues have been harvested and are being processed for morphological analyses.

Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries. In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases. For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonic stem cells, which have acquired properties of motor neurons. During the second year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assessed after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recordings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been published. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also performed transplantations of embryonic human stem cell derived motor neurons into the rat spinal cord and demonstrated surgical feasibility as well as survival of large numbers of neurons in the rat spinal cord. Some of the transplanted cells also demonstrate anatomical markers for motor neurons after transplantation.

During the reporting period, we have contined to demonstrate that human embryonic stem cell derived motor neurons and motor neuron progenitors can be produced in vitro. These motor neurons and motor neuron progenitors are transplanted into the rat spinal cord after a lumbosacral ventral root avulsion injury and repair of injured roots in the form of surgical re-attachment of the roots to the spinal cord surface. The lumbosacral ventral root avulsion injury mimics cauda equina and conus medullaris forms of spinal cord injury, an underserved patient population with paralysis of the legs and loss of bladder and bowel funcion. In this clinically relevant injury and repair model in rats, we have during the past several months demonstrated that transplanted human embryonic stem cell-derived motor neurons and motor neuron progenitors are able to survive in the spinal cord of rats over extended periods of time with large numbers of neurons being detectable in the spinal cord grey matter at 1, 2, and 10 weeks after the injury, surgical root repair, and transplantation of the cells. The long term viability of translanted cells suggests integration of the transplanted cells in the host tissues. Some of the cells show expression of motor neuron markers, such as the transcription factor Hb9, as demonstrated by immunohistochemistry and light microscopy. Additional studies have been performed during this reporting period to address whether the transplanted cells may extend axons into the replanted lumbosacral ventral roots. Interestingly, many human axons were detected in the replanted ventral roots using immunohistochemitry and light microscopy for the detection of human processes. Additional immunohistochemistry demonstrated that these processes contained neurofilaments, which are characteristic for axons. In control experiments, we showed that avulsed roots, which had not been replanted into the spinal cord, did not exhibit any human axons. As expected, surgical reconnection of lesioned ventral roots to the spinal cord is needed in order for the axons of the transplanted human embryonic stem cell derived motor neurons and motor neuron progenitors to be extended into avulsed ventral roots. Furthermore, in a series of sham operated animals without ventral root lesions, human motor neurons and motor neuron progenitors were also transplanted into the rat spinal cord. Interestingly, the transplanted human motor neurons and motor neuron progenitors were here also able to extend axons into ventral roots, even though the ventral roots had never been lesions. We conclude that transplanted human embryonic stem cell derived motor neurons are capable of extending axons into both intact ventrl roots and into ventral roots, which had been avulsed and surgically reattached to the spinal cord using a replantation procedure. In functional studies, we have performed urodynamic studies and voiding behavioral studies in rats after the transplantation of human embryonic stem cell derived motor neurons and motor neuron progenitors. These studies are still ongoing with additional experiments being performed. However, preliminary studies suggest that the combination of acute repair of avulsed ventral roots and cell transplantation results in a gradual improvement of voiding reflexes. Ongoing studies are addressing the relative contribution that may be provided by the replantation of avulsed ventral roots and by the transplantation of human motor neurons and motor neuron progenitors into the rat spinal cord.