Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Injured adult neurons regress to an embryonic transcriptional growth state.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.

Introduction for Stem Cell-Based Therapy for Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) are a group of neurological diseases caused by the progressive degeneration of neurons and glial cells in the brain and spinal cords. Usually there is a selective loss of specific neuronal cells in a restricted brain area from any neurodegenerative diseases, such as dopamine (DA) neuron death in Parkinson disease (PD) and motor neuron loss in amyotrophic lateral sclerosis (ALS), or a widespread degeneration affecting many types of neurons in Alzheimer's disease (AD). As there is no effective treatment to stop the progression of these neurodegenerative diseases, stem cell-based therapies have provided great potentials for these disorders. Currently transplantation of different stem cells or their derivatives has improved neural function in animal models of neurodegenerative diseases by replacing the lost neural cells, releasing cytokines, modulation of inflammation, and mediating remyelination. With the advance in somatic cell reprogramming to generate induced pluripotent stem cells (iPS cells) and directly induced neural stem cells or neurons, pluripotent stem cell can be induced to differentiate to any kind of neural cells and overcome the immune rejection of the allogeneic transplantation. Recent studies have proved the effectiveness of transplanted stem cells in animal studies and some clinical trials on patients with NDs. However, some significant hurdles need to be resolved before these preclinical results can be translated to clinic. In particular, we need to better understand the molecular mechanisms of stem cell transplantation and develop new approaches to increase the directed neural differentiation, migration, survival, and functional connections of transplanted stem cells in the pathological environment of the patient's central nerve system.

Stem Cell Transplantation for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a motor neuronal degeneration disease, in which the death of motor neurons causes lost control of voluntary muscles. The consequence is weakness of muscles with a wide range of disabilities and eventually death. Most patients died within 5 years after diagnosis, and there is no cure for this devastating neurodegenerative disease up to date. Stem cells, including non-neural stem cells and neural stem cells (NSCs) or neural progenitor cells (NPCs), are very attractive cell sources for potential neuroprotection and motor neuron replacement therapy which bases on the idea that transplant-derived and newly differentiated motor neurons can replace lost motor neurons to re-establish voluntary motor control of muscles in ALS. Our recent studies show that transplanted NSCs or NPCs not only survive well in injured spinal cord, but also function as neuronal relays to receive regenerated host axonal connection and extend their own axons to host for connectivity, including motor axons in ventral root. This reciprocal connection between host neurons and transplanted neurons provides a strong rationale for neuronal replacement therapy for ALS to re-establish voluntary motor control of muscles. In addition, a variety of new stem cell resources and the new methodologies to generate NSCs or motor neuron-specific progenitor cells have been discovered and developed. Together, it provides the basis for motor neuron replacement therapy with NSCs or NPCs in ALS.

Future Challenges and Perspectives for Stem Cell Therapy of Neurodegenerative Diseases.

Stem cell-based therapy has shown exciting efficacy in pre-clinical studies on different neurodegenerative diseases (NDs). However, no clinically applicable stem-cell-derived neurons are available to the patients with NDs. There exist some obstacles associated with stem cell therapy, which need to be overcome in future clinical studies. In this chapter, more challenges and new strategies will be explored to accelerate the clinical translation of a human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC)-derived neural cell product to patients with NDs.

Stem Cells Therapy for Multiple Sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) associated with inflammatory plaques of white matter demyelination, oligodendrocyte destruction, reactive gliosis and axonal degeneration. In this chapter, we first review the pathological process of axonal degeneration in MS and discuss how these changes cause clinical symptoms of MS. We then discuss the pharmacological treatment to improve the clinical symptoms. Finally, we highlight how the autologous hematopoietic stem cell transplantation (AHSCT) can be effective for aggressive MS patients, who fail to respond to drug therapies, and also propose the future challenges of AHSCT.

Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury.

Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system.

Partial restoration of cardiovascular function by embryonic neural stem cell grafts after complete spinal cord transection.

High-level spinal cord injury can lead to cardiovascular dysfunction, including disordered hemodynamics at rest and autonomic dysreflexia during noxious stimulation. To restore supraspinal control of sympathetic preganglionic neurons (SPNs), we grafted embryonic brainstem-derived neural stem cells (BS-NSCs) or spinal cord-derived neural stem cells (SC-NSCs) expressing green fluorescent protein into the T4 complete transection site of adult rats. Animals with injury alone served as controls. Implanting of BS-NSCs but not SC-NSCs resulted in recovery of basal cardiovascular parameters, whereas both cell grafts alleviated autonomic dysreflexia. Subsequent spinal cord retransection above the graft abolished the recovery of basal hemodynamics and reflexic response. BS-NSC graft-derived catecholaminergic and serotonergic neurons showed remarkable long-distance axon growth and topographical innervation of caudal SPNs. Anterograde tracing indicated growth of medullar axons into stem cell grafts and formation of synapses. Thus, grafted embryonic brainstem-derived neurons can act as functional relays to restore supraspinal regulation of denervated SPNs, thereby contributing to cardiovascular functional improvement.

Integration and long distance axonal regeneration in the central nervous system from transplanted primitive neural stem cells.

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.

A facilitatory role of astrocytes in axonal regeneration after acute and chronic spinal cord injury.

Neuroscience dogma avers that astrocytic "scars" inhibit axonal regeneration after spinal cord injury (SCI). A recent report suggested however that astrocytes form "borders" around lesions that are permissive rather than inhibitory to axonal growth. We now provide further evidence supporting a facilitatory role of astrocytes in axonal regeneration after SCI. First, even 6months after SCI, injured axons are retained within regions of densely reactive astrocytes, in direct contact with astrocyte processes without being repelled. Second, 6 month-delayed implants of neural stem cells extend axons into reactive astrocyte borders surrounding lesions, densely contacting astrocyte surfaces. Third, bioengineered hydrogels implanted into sites of SCI re-orient reactive astrocytic processes to align along the rostral-to-caudal spinal cord axis resulting in successful regeneration into the lesion/scaffold in close association with astrocytic processes. Fourth, corticospinal axons regenerate into neural progenitor cells implanted six months after injury in close association with host astrocytic processes. Thus, astrocytes do not appear to inhibit axonal regeneration, and the close association of newly growing axons with astrocytic processes suggests a facilitatory role in axonal regeneration.

A critical cell-intrinsic role for serum response factor in glial specification in the CNS.

Astrocytes and oligodendrocytes play crucial roles in nearly every facet of nervous system development and function, including neuronal migration, synaptogenesis, synaptic plasticity, and myelination. Previous studies have widely characterized the signaling pathways important for astrocyte differentiation and unveiled a number of transcription factors that guide oligodendrocyte differentiation in the CNS. However, the identities of the transcription factors critical for astrocyte specification in the brain remain unknown. Here we show that deletion of the stimulus-dependent transcription factor, serum response factor (SRF), in neural precursor cells (NPCs) (Srf-Nestin-cKO) results in nearly 60% loss in astrocytes and 50% loss in oligodendrocyte precursors at birth. Cultured SRF-deficient NPCs exhibited normal growth rate and capacity to self-renew. However, SRF-deficient NPCs generated fewer astrocytes and oligodendrocytes in response to several lineage-specific differentiation factors. These deficits in glial differentiation were rescued by ectopic expression of wild-type SRF in SRF-deficient NPCs. Interestingly, ectopic expression of a constitutively active SRF (SRF-VP16) in NPCs augmented astrocyte differentiation in the presence of pro-astrocytic factors. However, SRF-VP16 expression in NPCs had an inhibitory effect on oligodendrocyte differentiation. In contrast, mice carrying conditional deletion of SRF in developing forebrain neurons (Srf-NEX-cKO) did not exhibit any deficits in astrocytes in the brain. Together, our observations suggest that SRF plays a critical cell-autonomous role in NPCs to regulate astrocyte and oligodendrocyte specification in vivo and in vitro.

Motor axonal regeneration after partial and complete spinal cord transection.

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.

Adaptation of a cervical bilateral contusive spinal cord injury for study of skilled forelimb function.

We present an updated, clinically relevant model of moderately severe bilateral cervical level 6 contusive spinal cord injury (SCI) in the rat. This model is more clinically relevant than previous models due it its severity, yet animals readily survive the lesion. The C6 bilateral lesion is administered to Fischer 344 rats using the Infinite Horizons impactor adjusted to a 200 kdyne force with a 3.5 mm impactor head. The lesion results in loss of 60 ± 10% of the spinal cord area, including virtually the entire dorsal half of the spinal cord and complete interruption of the main corticospinal tract. Skilled forelimb performance declines by 60 ± 10% compared to the pre-operative baseline and deficits are sustained over time. This model is a substantial step closer to mimicking the most common level (cervical) and more severe form of SCI in humans and should provide a superior tool for assessing the likelihood that experimental interventions may promote motor recovery after SCI in humans.

BDNF guides neural stem cell-derived axons to ventral interneurons and motor neurons after spinal cord injury.

Neural stem cells (NSCs) implanted into sites of spinal cord injury (SCI) extend very large numbers of new axons over very long distances caudal to the lesion site, and support partial functional recovery. Newly extending graft axons distribute throughout host gray and white matter caudal to the injury. We hypothesized that provision of trophic gradients caudal to the injury would provide neurotrophic guidance to newly extending graft-derived axons to specific intermediate and ventral host gray matter regions, thereby potentially further improving neural relay formation. Immunodeficient rats underwent C5 lateral hemisection lesions, following by implants of human NSC grafts two weeks later. After an additional two weeks, animals received injections of AAV2-BDNF expressing vectors three spinal segments (9 mm) caudal to the lesion in host ventral and intermediate gray matter. After 2 months additional survival, we found a striking, 5.5-fold increase in the density of human axons innervating host ventral gray matter (P < 0.05) and 2.7-fold increase in intermediate gray matter (P < 0.01). Moreover, stem cell-derived axons formed a substantially greater number of putative synaptic connections with host motor neurons (P < 0.01). Thus, trophic guidance is an effective means of enhancing and guiding neural stem cell axon growth after SCI and will be used in future experiments to determine whether neural relay formation and functional outcomes can be improved.

Serum response factor is required for cortical axon growth but is dispensable for neurogenesis and neocortical lamination.

Previous studies have shown that neuron-specific deletion of serum response factor (SRF) results in deficits in tangential cell migration, guidance-dependent circuit assembly, activity-dependent gene expression, and synaptic plasticity in the hippocampus. Furthermore, SRF deletion in mouse embryonic stem cells causes cell death in vitro. However, the requirement of SRF for early neuronal development including neural stem cell homeostasis, neurogenesis, and axonal innervations remains unknown. Here, we report that SRF is critical for development of major axonal tracts in the forebrain. Conditional mutant mice lacking SRF in neural progenitor cells (Srf-Nestin-cKO) exhibit striking deficits in cortical axonal projections including corticostriatal, corticospinal, and corticothalamic tracts, and they show a variable loss of the corpus callosum. Neurogenesis and interneuron specification occur normally in the absence of SRF and the deficits in axonal projections were not due to a decrease or loss in cell numbers. Radial migration of neurons and neocortical lamination were also not affected. No aberrant cell death was observed during development, whereas there was an increase in the number of proliferative cells in the ventricular zone from embryonic day 14 to day 18. Similar axonal tract deficits were also observed in mutant mice lacking SRF in the developing excitatory neurons of neocortex and hippocampus (Srf-NEX-cKO). Together, these findings suggest distinct roles for SRF during neuronal development; SRF is specifically required in a cell-autonomous manner for axonal tract development but is dispensable for cell survival, neurogenesis, neocortical lamination, and neuronal differentiation.

PROSESS: a protein structure evaluation suite and server.

PROSESS (PROtein Structure Evaluation Suite and Server) is a web server designed to evaluate and validate protein structures generated by X-ray crystallography, NMR spectroscopy or computational modeling. While many structure evaluation packages have been developed over the past 20 years, PROSESS is unique in its comprehensiveness, its capacity to evaluate X-ray, NMR and predicted structures as well as its ability to evaluate a variety of experimental NMR data. PROSESS integrates a variety of previously developed, well-known and thoroughly tested methods to evaluate both global and residue specific: (i) covalent and geometric quality; (ii) non-bonded/packing quality; (iii) torsion angle quality; (iv) chemical shift quality and (v) NOE quality. In particular, PROSESS uses VADAR for coordinate, packing, H-bond, secondary structure and geometric analysis, GeNMR for calculating folding, threading and solvent energetics, ShiftX for calculating chemical shift correlations, RCI for correlating structure mobility to chemical shift and PREDITOR for calculating torsion angle-chemical shifts agreement. PROSESS also incorporates several other programs including MolProbity to assess atomic clashes, Xplor-NIH to identify and quantify NOE restraint violations and NAMD to assess structure energetics. PROSESS produces detailed tables, explanations, structural images and graphs that summarize the results and compare them to values observed in high-quality or high-resolution protein structures. Using a simplified red-amber-green coloring scheme PROSESS also alerts users about both general and residue-specific structural problems. PROSESS is intended to serve as a tool that can be used by structure biologists as well as database curators to assess and validate newly determined protein structures. PROSESS is freely available at http://www.prosess.ca.

Motor neuron replacement therapy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a motor neuron degenerative disease that is also known as Lou Gehrig's disease in the United States, Charcot's disease in France, and motor neuron disease in the UK. The loss of motor neurons causes muscle wasting, paralysis, and eventually death, which is commonly related to respiratory failure, within 3-5 years after onset of the disease. Although there are a limited number of drugs approved for amyotrophic lateral sclerosis, they have had little success at treating the associated symptoms, and they cannot reverse the course of motor neuron degeneration. Thus, there is still a lack of effective treatment for this debilitating neurodegenerative disorder. Stem cell therapy for amyotrophic lateral sclerosis is a very attractive strategy for both basic and clinical researchers, particularly as transplanted stem cells and stem cell-derived neural progenitor/precursor cells can protect endogenous motor neurons and directly replace the lost or dying motor neurons. Stem cell therapies may also be able to re-establish the motor control of voluntary muscles. Here, we review the recent progress in the use of neural stem cells and neural progenitor cells for the treatment of amyotrophic lateral sclerosis. We focus on MN progenitor cells derived from fetal central nervous system tissue, embryonic stem cells, and induced pluripotent stem cells. In our recent studies, we found that transplanted human induced pluripotent stem cell-derived motor neuron progenitors survive well, differentiate into motor neurons, and extend axons into the host white matter, not only in the rostrocaudal direction, but also along motor axon tracts towards the ventral roots in the immunodeficient rat spinal cord. Furthermore, the significant motor axonal extension after neural progenitor cell transplantation in amyotrophic lateral sclerosis models demonstrates that motor neuron replacement therapy could be a promising therapeutic strategy for amyotrophic lateral sclerosis, particularly as a variety of stem cell derivatives, including induced pluripotent stem cells, are being considered for clinical trials for various diseases.

Rehabilitation combined with neural progenitor cell grafts enables functional recovery in chronic spinal cord injury.

We reported previously that neural progenitor cell (NPC) grafts form neural relays across sites of subacute spinal cord injury (SCI) and support functional recovery. Here, we examine whether NPC grafts after chronic delays also support recovery and whether intensive rehabilitation further enhances recovery. One month after severe bilateral cervical contusion, rats received daily intensive rehabilitation, NPC grafts, or both rehabilitation and grafts. Notably, only the combination of rehabilitation and grafting significantly improved functional recovery. Moreover, improved functional outcomes were associated with a rehabilitation-induced increase in host corticospinal axon regeneration into grafts. These findings identify a critical and synergistic role of rehabilitation and neural stem cell therapy in driving neural plasticity to support functional recovery after chronic and severe SCI.