Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF

Oxygen Tension Modulates Neurite Outgrowth in PC12 Cells Through A Mechanism Involving HIF and VEGF

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1α stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair

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

Critical Role of PI3K/Akt/GSK3β in Motoneuron Specification from Human Neural Stem Cells in Response to FGF2 and EGF

Fibroblast growth factor (FGF) and epidermal growth factor (EGF) are critical for the development of the nervous system. We previously discovered that FGF2 and EGF had opposite effects on motor neuron differentiation from human fetal neural stem cells (hNSCs), but the underlying mechanisms remain unclear. Here, we show that FGF2 and EGF differentially affect the temporal patterns of Akt and glycogen synthase kinase 3 beta (GSK3β) activation. High levels of phosphatidylinositol 3-kinase (PI3K)/Akt activation accompanied with GSK3β inactivation result in reduction of the motor neuron transcription factor HB9. Inhibition of PI3K/Akt by chemical inhibitors or RNA interference or overexpression of a constitutively active form of GSK3β enhances HB9 expression. Consequently, PI3K inhibition increases hNSCs differentiation into HB9+/microtubule-associated protein 2 (MAP2)+ motor neurons in vitro. More importantly, blocking PI3K not only enhances motor neuron differentiation from hNSCs grafted into the ventral horn of adult rat spinal cords, but also permits ectopic generation of motor neurons in the dorsal horn by overriding environmental influences. Our data suggest that FGF2 and EGF affect the motor neuron fate decision in hNSCs differently through a fine tuning of the PI3K/AKT/GSK3β pathway, and that manipulation of this pathway can enhance motor neuron generation

Effects of Dibutyryl Cyclic-AMP on Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells Transplanted into Spinal Cord Injured Rats

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers

Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal coRd Injury NOD-scid Mouse Model

Traumatic spinal cord injury (SCI) results in partial or complete paralysis and is characterized by a loss of neurons and oligodendrocytes, axonal injury, and demyelination/dysmyelination of spared axons. Approximately 1,250,000 individuals have chronic SCI in the U.S.; therefore treatment in the chronic stages is highly clinically relevant. Human neural stem cells (hCNS-SCns) were prospectively isolated based on fluorescence-activated cell sorting for a CD133(+) and CD24(-/lo) population from fetal brain, grown as neurospheres, and lineage restricted to generate neurons, oligodendrocytes and astrocytes. hCNS-SCns have recently been transplanted sub-acutely following spinal cord injury and found to promote improved locomotor recovery. We tested the ability of hCNS-SCns transplanted 30 days post SCI to survive, differentiate, migrate, and promote improved locomotor recovery.hCNS-SCns were transplanted into immunodeficient NOD-scid mice 30 days post spinal cord contusion injury. hCNS-SCns transplanted mice demonstrated significantly improved locomotor recovery compared to vehicle controls using open field locomotor testing and CatWalk gait analysis. Transplanted hCNS-SCns exhibited long-term engraftment, migration, limited proliferation, and differentiation predominantly to oligodendrocytes and neurons. Astrocytic differentiation was rare and mice did not exhibit mechanical allodynia. Furthermore, differentiated hCNS-SCns integrated with the host as demonstrated by co-localization of human cytoplasm with discrete staining for the paranodal marker contactin-associated protein.The results suggest that hCNS-SCns are capable of surviving, differentiating, and promoting improved locomotor recovery when transplanted into an early chronic injury microenvironment. These data suggest that hCNS-SCns transplantation has efficacy in an early chronic SCI setting and thus expands the "window of opportunity" for intervention

Mesenchymal Stem Cell Graft Improves Recovery after Spinal Cord Injury in Adult Rats through Neurotrophic and Pro-Angiogenic Actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue