IntroductionIntraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury.MethodsThree-month-old female Sprague–Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies.ResultsIntraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons.ConclusionsPeri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.
BackgroundDue to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.Methodology/Principal FindingsHESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.Conclusions/SignificanceThe application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.
In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184+ population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/ DCX-immunoreactive neurons were seen with extensive DCX + processes. At survival intervals of 4-8 weeks, hNSE + neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA + cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FACsorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.
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