Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recovery after cervical spinal cord injury.

Evidence that cell transplants can improve recovery outcomes in spinal cord injury (SCI) models substantiates treatment strategies involving cell replacement for humans with SCI. Most pre-clinical studies of cell replacement in SCI examine thoracic injury models. However, as most human injuries occur at the cervical level, it is critical to assess potential treatments in cervical injury models and examine their effectiveness using at-level histological and functional measures. To directly address cervical SCI, we used a C5 midline contusion injury model and assessed the efficacy of a candidate therapeutic for thoracic SCI in this cervical model. The contusion generates reproducible, bilateral movement and histological deficits, although a number of injury parameters such as acute severity of injury, affected gray-to-white matter ratio, extent of endogenous remyelination, and at-level locomotion deficits do not correspond with these parameters in thoracic SCI. On the basis of reported benefits in thoracic SCI, we transplanted human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into this cervical model. hESC-derived OPC transplants attenuated lesion pathogenesis and improved recovery of forelimb function. Histological effects of transplantation included robust white and gray matter sparing at the injury epicenter and, in particular, preservation of motor neurons that correlated with movement recovery. These findings further our understanding of the histopathology and functional outcomes of cervical SCI, define potential therapeutic targets, and support the use of these cells as a treatment for cervical SCI.

The extent of myelin pathology differs following contusion and transection spinal cord injury.

Demyelination is a prominent feature of spinal cord injury (SCI) and is followed by incomplete remyelination, which may contribute to physiological impairment. Demyelination has been documented in several species including humans, but the extent of demyelination and its functional consequence remain unknown. In this report, we document and compare the extent of tissue pathology, white matter apoptosis, demyelination, and remyelination 2 months following injury in rat contusion and transection models of SCI. Moreover, we document and compare the macrophage response 3 and 14 days post contusion and transection SCI. Contusion injury resulted in widespread tissue pathology, white matter apoptosis, demyelination, incomplete remyelination, and robust macrophage response extending several millimeters cranial and caudal to the epicenter of injury. In contrast, transection injury resulted in focal tissue pathology with white matter apoptosis, demyelination, incomplete remyelination, and robust macrophage response at the epicenter of injury, and little pathologic features at a distance from the epicenter of injury, as indicated by the lack of apoptosis and demyelination. These data indicate for the first time that myelin pathology differs substantially following contusion and transection SCI.

Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.

Motor neuron loss is characteristic of many neurodegenerative disorders and results in rapid loss of muscle control, paralysis, and eventual death in severe cases. In order to investigate the neurotrophic effects of a motor neuron lineage graft, we transplanted human embryonic stem cell-derived motor neuron progenitors (hMNPs) and examined their histopathological effect in three animal models of motor neuron loss. Specifically, we transplanted hMNPs into rodent models of SMA (Δ7SMN), ALS (SOD1 G93A), and spinal cord injury (SCI). The transplanted cells survived and differentiated in all models. In addition, we have also found that hMNPs secrete physiologically active growth factors in vivo, including NGF and NT-3, which significantly enhanced the number of spared endogenous neurons in all three animal models. The ability to maintain dying motor neurons by delivering motor neuron-specific neurotrophic support represents a powerful treatment strategy for diseases characterized by motor neuron loss.

Derivation of high purity neuronal progenitors from human embryonic stem cells.

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.

Myelin pathogenesis and functional deficits following SCI are age-associated.

Most spinal cord injuries (SCI) occur in young adults. In the past few decades however, the average age at time of SCI and the percentage of injuries in persons over the age of 60 have increased. Studies have shown that there is an age-associated delay in the rate of remyelination following toxin-induced demyelination of the spinal cord, suggesting that there may be an age-associated difference in regenerative efficiency. Here we examine for the first time locomotor recovery, bladder recovery, and myelin pathology in young (3 months), aged (12 months), and geriatric (24 months) female rats following contusion SCI. Our assessments indicate that aged and geriatric rats have a delayed rate of locomotor recovery following contusion SCI as compared to young rats. Additionally, aged and geriatric rats have significantly slower bladder recovery as compared to young rats. Examination of myelin pathology reveals that aged and geriatric rats have significantly greater area of pathology and amount of demyelination, as well as significantly less remyelination as compared to young rats following contusion SCI. These data are the first to indicate that there is an age-associated decline in the rate and extent of both locomotor and bladder recovery following contusion SCI, and that age adversely affects the degree of general pathology, demyelination, and remyelination that accompanies contusion SCI.

Voluntary running attenuates age-related deficits following SCI.

Over the past few decades, the average age at time of spinal cord injury (SCI) has increased. Here we examined locomotor recovery and myelin pathology in both young and aged adult rats following contusion SCI. Our assessment indicates that the rate of locomotor recovery following SCI is significantly delayed in aged rats as compared to young rats, and is associated with a greater degree of pathology and demyelination. Additionally, we examined the effect of voluntary exercise, pre- and post-injury, on locomotor recovery and myelin pathology following contusion SCI. Our data indicate that exercise improves the locomotor recovery of injured aged rats such that it is comparable to the recovery rate of injured young rats, and is associated with a decreased area of pathology and amount of demyelination. Interestingly, the rate of locomotor recovery and myelin pathology in the aged exercised rats was similar to that of the young sedentary rats after injury, indicating that exercise attenuates the delayed recovery of function and associated histopathology in aged rats. These data indicate that there is an age-related delay in locomotor recovery following SCI, and an age-related increase in histopathology following SCI. Importantly, our data indicate that exercise attenuates these age-related deficits following SCI.

Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis.

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.

Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.