Transplantation of neural progenitors remains a promising therapeutic approach to spinal cord injury (SCI), but the anatomical and functional evaluation of their effects is complex, particularly when using human cells. We investigated the outcome of transplanting human glial-restricted progenitors (hGRP) and astrocytes derived from hGRP (hGDA) in spinal cord contusion with respect to cell fate and host response using athymic rats to circumvent xenograft immune issues. Nine days after injury hGRP, hGDA, or medium were injected into the lesion center and rostral and caudal to the lesion, followed by behavioral testing for 8 weeks. Both hGRP and hGDA showed robust graft survival and extensive migration. The total number of cells increased 3.5-fold for hGRP, and twofold for hGDA, indicating graft expansion, but few proliferating cells remained by 8 weeks. Grafted cells differentiated into glia, predominantly astrocytes, and few remained at progenitor state. About 80% of grafted cells around the injury were glial fibrillary acidic protein (GFAP)-positive, gradually decreasing to 40-50% at a distance of 6 mm. Conversely, there were few graft-derived oligodendrocytes at the lesion, but their numbers increased away from the injury to 30-40%. Both cell grafts reduced cyst and scar formation at the injury site compared to controls. Microglia/macrophages were present at and around the lesion area, and axons grew along the spared tissue with no differences among groups. There were no significant improvements in motor function recovery as measured by the Basso, Beattie, and Bresnahan (BBB) scale and grid tests in all experimental groups. Cystometry revealed that hGRP grafts attenuated hyperactive bladder reflexes. Importantly, there was no increased sensory or tactile sensitivity associated with pain, and the hGDA group showed sensory function returning to normal. Although the improved lesion environment was not sufficient for robust functional recovery, the permissive properties and lack of sensory hypersensitivity indicate that human GRP and astrocytes remain promising candidates for therapy after SCI.

The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia regulate synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. Astroglial mechanisms underlying this essential neuron-glial communication are not known. We now show that presynaptic terminals regulate astroglial synaptic functions, GLT1/EAAT2, via kappa B-motif binding phosphoprotein (KBBP), the mouse homolog of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the GLT1/EAAT2 promoter. Neuron-stimulated KBBP is required for GLT1/EAAT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling by corticospinal tract transection, ricin-induced motor neuron death, or neurodegeneration in amyotrophic lateral sclerosis all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.

The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia regulate synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. Astroglial mechanisms underlying this essential neuron-glial communication are not known. We now show that presynaptic terminals regulate astroglial synaptic functions, GLT1/EAAT2, via kappa B-motif binding phosphoprotein (KBBP), the mouse homolog of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the GLT1/EAAT2 promoter. Neuron-stimulated KBBP is required for GLT1/EAAT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling by corticospinal tract transection, ricin-induced motor neuron death, or neurodegeneration in amyotrophic lateral sclerosis all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.

OBJECT Stem cell therapy has been shown to have considerable therapeutic potential for spinal cord injuries (SCIs); however, most experiments in animals have been performed by injecting cells directly into the injured parenchyma. This invasive technique compromises the injured spinal cord, although it delivers cells into the hostile environment of the acutely injured cord. In this study, the authors tested the possibility of delivering stem cells to injured spinal cord by using three different minimally invasive techniques. METHODS Bone marrow stromal cells (BMSCs) are clinically attractive because they have shown therapeutic potential in SCI and can be obtained in patients at the bedside, raising the possibility of autologous transplantation. In this study transgenically labeled cells were used for transplantation, facilitating posttransplantation tracking. Inbred Fisher-344 rats received partial cervical hemisection injury, and 2 x 10(6) BMSCs were intravenously, intraventricularly, or intrathecally transplanted 24 hours later via lumbar puncture (LP). The animals were killed 3, 10, or 14 days posttransplantation, and tissue samples were submitted to histochemical and immunofluorescence analyses. For additional comparison and validation, lineage restricted neural precursor (LRNP) cells obtained from E13.5 rat embryos were transplanted via LP, and these findings were also analyzed. CONCLUSIONS Both BMSCs and LRNP cells home toward injured spinal cord tissues. The use of LP and intraventricular routes allows more efficient delivery of cells to the injured cord compared with the intravenous route. Stem cells delivered via LP for treatment of SCI may potentially be applicable in humans after optimal protocols and safety profiles are established in further studies.

Talpha-1 tubulin promoter-driven EYFP expression is seen in murine neurons born as early as E9.5. Double labeling with markers for stem cells (Sox 1, Sox 2, nestin), glial progenitors (S100beta, NG2, Olig2), and neuronal progenitors (doublecortin, betaIII-tubulin, PSA-NCAM) show that Talpha-1 tubulin expression is limited to early born neurons. BrdU uptake and double labeling with neuronal progenitor markers in vivo and in vitro show that EYFP-expressing cells are postmitotic and Talpha-1 tubulin EYFP precedes the expression of MAP-2 and NeuN, and follows the expression of PSA-NCAM, doublecortin (Dcx), and betaIII-tubulin. Talpha-1 tubulin promoter-driven EYFP expression is transient and disappears in most neurons by P0. Persistent EYFP expression is mainly limited to scattered cells in the subventricular zone (SVZ), rostral migratory stream, and hippocampus. However, there are some areas that continue to express Talpha-1 tubulin in the adult without apparent neurogenesis. The number of EYFP-expressing cells declines with age indicating that Talpha-1 tubulin accurately identifies early born postmitotic neurons throughout development but less clearly in the adult. Assessment of neurogenesis after stab wound injuries in the cortex, cerebellum and spinal cord of adult animals shows no neurogenesis in most areas with an increase in BrdU incorporation in glial and other non neuronal populations. An up-regulation of Talpha-1 tubulin can be seen in certain areas unaccompanied by new neurogenesis. Our results suggest that even if stem cells proliferate their ability to generate neurons is limited and caution is warranted in attributing increased BrdU incorporation to stem cells or cells fated to be neurons even in neurogenic areas.