Neural Stem/Progenitor Cells Derived from the Embryonic Dorsal Telencephalon of D6/GFP Mice Differentiate Primarily into Neurons After Transplantation into a Cortical Lesion

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na⁺-currents and K _A- and K _DR currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.     

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.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

3D Differentiation of Neural Stem Cells in Macroporous Photopolymerizable Hydrogel Scaffolds

Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm², 10 wt%, 6330±1160 µm², 20 wt%, 7600±1550 µm²) compared with controls (0 wt%, 3150±220 µm²). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Macrophage Migration Inhibitory Factor Promotes Proliferation and Neuronal Differentiation of Neural Stem/Precursor Cells through Wnt/β-Catenin Signal Pathway

Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.     

Viral infection and neural stem/progenitor cell's fate: implications in brain development and neurological disorders

Viral infections in the prenatal (during pregnancy) and perinatal period have been a common cause of brain malformation. Besides the immediate neurological dysfunctions, virus infections may critically affect CNS development culminating in long-term cognitive deficits. Most of these neurotropic viruses are most damaging at a critical stage of the host, when the brain is in a dynamic stage of development. The neuropathology can be attributed to the massive neuronal loss induced by the virus as well as lack of CNS repair owing to a deficit in the neural stem/progenitor cell (NSPC) pool or aberrant formation of new neurons from NSPCs. Being one of the mitotically active populations in the post natal brain, the NSPCs have emerged as the potential targets of neurotropic viruses. The NSPCs are self-renewing and multipotent cells residing in the neurogenic niches of the brain, and, therefore, hampering the developmental fate of these cells may adversely affect the overall neurogenesis pattern. A number of neurotropic viruses utilize NSPCs as their cellular reservoirs and often establish latent and persistent infection in them. Both HIV and Herpes virus infect NSPCs over long periods of time and reactivation of the virus may occur later in life. The virus infected NSPCs either undergoes cell cycle arrest or impaired neuronal or glial differentiation, all of which leads to impaired neurogenesis. The disturbances in neurogenesis and CNS development following neurotropic virus infections have direct implications in the viral pathogenesis and long-term neurobehavioral outcome in infected individuals.     

Tenascin-R distinct domains modulate migration of neural stem/progenitor cells in vitro

Extracellular matrix (ECM) molecules constitute a "niche" that modulates the migration, proliferation, and differentiation of neural stem/progenitor cells (NSPCs). The glycoprotein Tenascin-R (TN-R) is an ECM molecule, comprising multiple domains. Either the whole TN-R molecule or its distinct domains has been demonstrated to play a very important role in the developing central nervous system. However, little is known about the effect of the TN-R domain on NSPCs, especially NSPC migration. In the present study, we first show that both TN-R domains epidermal growth factor-like repeat (EGFL) and fibronectin type III (FN)6-8 can inhibit the NSPCs migration from neurospheres in vitro. Furthermore, both the EGFL and FN6-8 domains affect the distribution of neurons generated from neurospheres, indicating that EGFL and FN6-8 domains inhibit the motility of neurons generated from neurospheres. These results suggest that TN-R has an inhibitory effect on NSPCs migration.     

Neuronal roles of the integrin-associated protein (IAP/CD47) in developing cortical neurons

Little is known about the role of the integrin-associated protein (IAP, or CD47) in neuronal development and its function in the central nervous system. We investigated neuronal responses in IAP-overexpressing cortical neurons using a virus-gene transfer system. We found that dendritic outgrowth was significantly enhanced in IAP (form 4)-transfected neurons. Furthermore, synaptic proteins including synaptotagmin, syntaxin, synapsin I, and SNAP25 (25-kDa synaptosomal associated protein) were up-regulated. In accordance with this finding, the release of the excitatory transmitter glutamate and the frequencies of Ca2+ oscillations (glutamate-mediated synaptic transmission) were increased. Interestingly, the overexpression of IAP activated mitogen-activated protein kinase (MAPK), and this activation was required for the IAP-dependent biological effects. After down-regulation of the endogenous IAP by small interfering RNA, MAPK activity, synaptic protein levels, and glutamate release decreased. These observations suggest that the IAP plays important roles in dendritic outgrowth and synaptic transmission in developing cortical neurons through the activation of MAPK.     

Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca²⁺ fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.     

Expression of CD47/integrin-associated protein induces death of cultured cerebral cortical neurons

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is involved in brain-derived neurotrophic factor (BDNF)-promoted neuronal cell survival. Here, we report the apoptosis-inducing effect of CD47/integrin-associated protein (IAP), the heterophilic binding partner of BIT/SHPS-1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT-mediated biotin-dUTP nick-end labelling (TUNEL)-positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad-spectrum caspase inhibitor Z-VAD-fmk. Furthermore, we observed that co-expression of CD47/IAP with BIT/SHPS-1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase-dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP-induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS-1 and by BDNF.     

Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα

Background

The identification of factors that compromise neurogenesis is aimed at improving stem cell-based approaches in the field of regenerative medicine. Interferon gamma (IFNγ) is a main pro-inflammatory cytokine and up-regulated during several neurological diseases. IFNγ is generally thought to beneficially enhance neurogenesis from fetal or adult neural stem/precursor cells (NSPCs).

Results

We now provide direct evidence to the contrary that IFNγ induces a dysfunctional stage in a substantial portion of NSPC-derived progeny in vitro characterized by simultaneous expression of glial fibrillary acid protein (GFAP) and neuronal markers, an abnormal gene expression and a functional phenotype neither typical for neurons nor for mature astrocytes. Dysfunctional development of NSPCs under the influence of IFNγ was finally demonstrated by applying the microelectrode array technology. IFNγ exposure of NSPCs during an initial 7-day proliferation period prevented the subsequent adequate differentiation and formation of functional neuronal networks.

Conclusions

Our results show that immunocytochemical analyses of NSPC-derived progeny are not necessarily indicating the correct cellular phenotype specifically under inflammatory conditions and that simultaneous expression of neuronal and glial markers rather point to cellular dysregulation. We hypothesize that inhibiting the impact of IFNγ on NSPCs during neurological diseases might contribute to effective neurogenesis and regeneration.     

Region-specific Differentiation Potential of Adult Rat Spinal Cord Neural Stem/Precursors and Their Plasticity in Response to In Vitro Manipulation

This study characterized the differentiation of neural stem/precursor cells (NSPCs) isolated from different levels of the spinal cord (cervical vs lumbar cord) and different regions along the neuraxis (brain vs cervical spinal cord) of adult male Wistar enhanced green fluorescent protein rats. The differentiation of cervical spinal cord NSPCs was further examined after variation of time in culture, addition of growth factors, and changes in cell matrix and serum concentration. Brain NSPCs did not differ from cervical cord NSPCs in the percentages of neurons, astrocytes, or oligodendrocytes but produced 26.9% less radial glia. Lumbar cord NSPCs produced 30.8% fewer radial glia and 6.9% more neurons compared with cervical cord NSPCs. Spinal cord NSPC differentiation was amenable to manipulation by growth factors and changes in in vitro conditions. This is the first study to directly compare the effect of growth factors, culturing time, serum concentration, and cell matrix on rat spinal cord NSPCs isolated, propagated, and differentiated under identical conditions. (J Histochem Cytochem 57:405–423, 2009)     

The neuronal differentiation microenvironment is essential for spinal cord injury repair

Spinal cord injury (SCI) often leads to substantial disability due to loss of motor function and sensation below the lesion. Neural stem cells (NSCs) are a promising strategy for SCI repair. However, NSCs rarely differentiate into neurons; they mostly differentiate into astrocytes because of the adverse microenvironment present after SCI. We have shown that myelin-associated inhibitors (MAIs) inhibited neuronal differentiation of NSCs. Given that MAIs activate epidermal growth factor receptor (EGFR) signaling, we used a collagen scaffold-tethered anti-EGFR antibody to attenuate the inhibitory effects of MAIs and create a neuronal differentiation microenvironment for SCI repair. The collagen scaffold modified with anti-EGFR antibody prevented the inhibition of NSC neuronal differentiation by myelin. After transplantation into completely transected SCI animals, the scaffold-linked antibodies induced production of nascent neurons from endogenous and transplanted NSCs, which rebuilt the neuronal relay by forming connections with each other or host neurons to transmit electrophysiological signals and promote functional recovery. Thus, a scaffold-based strategy for rebuilding the neuronal differentiation microenvironment could be useful for SCI repair.     

Biophysical characteristics reveal neural stem cell differentiation potential

Background

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.

Methodology/Principal Findings

We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.

Conclusions/Significance

We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.     

The on/off of Pax6 controls the tempo of neuronal differentiation in the developing spinal cord

During neurogenesis, complex networks of genes act sequentially to control neuronal differentiation. In the neural tube, the expression of Pax6, a paired-box-containing gene, just precedes the appearance of the first post-mitotic neurons. So far, its only reported function in the spinal cord is in specifying subsets of neurons. Here we address its possible function in controlling the balance between proliferation and commitment of neural progenitors. We report that increasing Pax6 level is sufficient to push neural progenitors toward cell cycle exit and neuronal commitment via Neurogenin 2 (Ngn2) upregulation. However, neuronal precursors maintaining Pax6(On) fail to perform neuronal differentiation. Conversely, turning off Pax6 function in these precursors is sufficient to provoke premature differentiation and the number of differentiated neurons depends of the amount of Pax6 protein. Moreover, we found that Pax6 expression involves negative feedback regulation by Ngn2 and this repression is critical for the proneural activity of Ngn2. We present a model in which the level of Pax6 activity first conditions the moment when a given progenitor will leave the cell cycle and second, the moment when a selected neuronal precursor will irreversibly differentiate.