Neural Progenitor Cell Cultures Research Articles

Potassium channel expression in adult murine neural progenitor cells

Neural progenitor cells (NPCs) are a source of new neurons and glia in the adult brain. Most NPCs reside in the forebrain subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus, where they contribute to plasticity in the adult brain. To use their potential for repair, it is essential to identify the molecules that regulate their growth, migration and differentiation. Potassium (K +) channels are promising molecule candidates for NPC regulation as they are important components of signal transduction and their diversity is ideal to cover the complex functions required for cell proliferation and differentiation. There is increasing evidence that K + channels influence cell growth and neurogenesis, however, very little is known regarding K + channel distribution in NPCs. We therefore explored the expression of a variety of voltage-gated (Kv), inwardly rectifying (Kir) and two-pore (K2P) K + channels in the SVZ of adult mice and in neurosphere cultures of NPCs during growth and differentiation. Immunocytochemical analysis revealed a differential expression pattern of K + channels in nestin + SVZ precursor cells, early SVZ doublecortin + neurons and (sub)ependymal cells. These findings were confirmed in neurosphere cultures at the protein and mRNA levels. The expression of some K + channel proteins, such as Kir4.1, Kir6.1, TREK1 or TASK1, suggests a role of K + channels in the complex regulation of NPC proliferation, maturation and differentiation.

Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination.

Methamphetamine (METH) abuse has reached epidemic proportions, and it has become increasingly recognized that abusers suffer from a wide range of neurocognitive deficits. Much previous work has focused on the deleterious effects of METH on mature neurons, but little is known about the effects of METH on neural progenitor cells (NPCs). It is now well established that new neurons are continuously generated from NPCs in the adult hippocampus, and accumulating evidence suggests important roles for these neurons in hippocampal-dependent cognitive functions. In a rat hippocampal NPC culture system, we find that METH results in a dose-dependent reduction of NPC proliferation, and higher concentrations of METH impair NPC survival. NPC differentiation, however, is not affected by METH, suggesting cell-stage specificity of the effects of METH. We demonstrate that the effects of METH on NPCs are, in part, mediated through oxidative and nitrosative stress. Further, we identify seventeen NPC proteins that are post-translationally modified via 3-nitrotyrosination in response to METH, using mass spectrometric approaches. One such protein was pyruvate kinase isoform M2 (PKM2), an important mediator of cellular energetics and proliferation. We identify sites of PKM2 that undergo nitrotyrosination, and demonstrate that nitration of the protein impairs its activity. Thus, METH abuse may result in impaired adult hippocampal neurogenesis, and effects on NPCs may be mediated by protein nitration. Our study has implications for the development of novel therapeutic approaches for METH-abusing individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired.

SDF-1α Secreted by Human CD133-Derived Multipotent Stromal Cells Promotes Neural Progenitor Cell Survival Through CXCR7

We recently reported that concentrated conditioned medium (CdM) from human CD133-derived bone marrow progenitor cells (CD133 CdM) was neuroprotective after stroke. Here we identify stromal-derived factor 1 alpha (SDF-1) as a potential neuroprotective candidate in CD133 CdM by interrogating the transcriptional responses of CD133-derived multipotent stromal cells (CD133dMSCs) after cell injection into the ischemic brain. Human SDF-1 mRNA was upregulated 79-fold by CD133dMSCs when injected into the stroke peri-infarct area compared with cells injected into the uninjured parenchyma of sham-operated animals. In cell protection assays, we replaced the typical growth medium in mouse neural progenitor cell (mNPC) cultures with serum-free CD133 CdM immediately before exposure to hypoxia (1% oxygen) for 48 h. CD133 CdM significantly increased the survival of mNPCs during hypoxia exposure and growth factor withdrawal. To determine whether MSC-secreted SDF-1 influenced mNPC survival, we used lentiviral short hairpin RNA against SDF1 (shSDF-1) to knockdown SDF-1 expression in CD133dMSCs. The CdM generated from shSDF-1-treated cells had a 94% decrease in secreted SDF-1 and was significantly less protective for mNPCs when compared with control CdM from CD133dMSCs transduced with scrambled short hairpin RNA. Pharmacological inhibition of the 2 known SDF-1 receptors, CXCR4 and CXCR7, revealed that only CXCR7 activity was functionally linked to survival signaling in mNPCs during hypoxia exposure. Treatment of mNPCs with CD133 CdM and CXCR7 inhibitor decreased mNPC viability by 36.5% ± 12.8% and decreased cell number by 21% ± 6.7% compared with dimethyl sulfoxide treated controls. These data indicate that SDF-1 is a key neuroprotective cytokine secreted by CD133dMSCs that protects mNPCs through CXCR7.

Seeding neural progenitor cells on silicon-based neural probes

Chronically implanted neural electrode arrays have the potential to be used as neural prostheses in patients with various neurological disorders. While these electrodes perform well in acute recordings, they often fail to function reliably in clinically relevant chronic settings because of glial encapsulation and the loss of neurons. Surface modification of these implants may provide a means of improving their biocompatibility and integration within host brain tissue. The authors proposed a method of improving the brain-implant interface by seeding the implant's surface with a layer of neural progenitor cells (NPCs) derived from adult murine subependyma. Neural progenitor cells may reduce the foreign body reaction by presenting a tissue-friendly surface and repair implant-induced injury and inflammation by releasing neurotrophic factors. In this study, the authors evaluated the growth and differentiation of NPCs on laminin-immobilized probe surfaces and explored the potential impact on transplant survival of these cells. Laminin protein was successfully immobilized on the silicon surface via covalent binding using silane chemistry. The growth, adhesion, and differentiation of NPCs expressing green fluorescent protein (GFP) on laminin-modified silicon surfaces were characterized in vitro by using immunocytochemical techniques. Shear forces were applied to NPC cultures in growth medium to evaluate their shearing properties. In addition, neural probes seeded with GFP-labeled NPCs cultured in growth medium for 14 days were implanted in murine cortex. The authors assessed the adhesion properties of these cells during implantation conditions. Moreover, the tissue response around NPC-seeded implants was observed after 1 and 7 days postimplantation. Significantly improved NPC attachment and growth was found on the laminin-immobilized surface compared with an unmodified control before and after shear force application. The NPCs grown on the laminin-immobilized surface showed differentiation potential similar to those grown on polylysine-treated well plates, as previously reported. Viable (still expressing GFP) NPCs were found on and in proximity to the neural implant after 1 and 7 days postimplantation. Preliminary examinations indicated that the probe's NPC coating might reduce the glial response at these 2 different time points. The authors' findings suggest that NPCs can differentiate and strongly adhere to laminin-immobilized surfaces, providing a stable matrix for these cells to be implanted in brain tissue on the neural probe's surface. In addition, NPCs were found to improve the astrocytic reaction around the implant site. Further in vivo work revealing the mechanisms of this effect could lead to improvement of biocompatibility and chronic recording performance of neural probes.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

BackgroundNolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown.ResultsHere we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development.ConclusionsNolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Impaired neurogenesis is an early event in the etiology of familial Alzheimer's disease in transgenic mice.

Formation of new neurons in the adult brain takes place in the subventricular zone and in the subgranule layer of the dentate gyrus throughout life. Neurogenesis is thought to play a role in hippocampus- and olfaction-dependent learning and memory. However, whether impairments in neurogenesis take place in learning and memory disorders, such as Alzheimer's disease, is yet to be established. Importantly, it remains to be elucidated whether neurogenic impairments play a role in the course of the disease or are the result of extensive neuropathology. We now report that transgenic mice harboring familial Alzheimer's disease-linked mutant APPswe/PS1DeltaE9 exhibit severe impairments in neurogenesis that are evident as early as 2 months of age. These mice exhibit a significant reduction in the proliferation of neural progenitor cells and their neuronal differentiation. Interestingly, levels of hyperphosphorylated tau, the cytotoxic precursor of the Alzheimer's disease hallmark neurofibrillary tangles, are particularly high in the neurogenic niches. Isolation of neural progenitor cells in culture reveals that APPswe/PS1DeltaE9-expressing neurospheres exhibit impaired proliferation and tau hyperphosphorylation compared with wildtype neurospheres isolated from nontransgenic littermates. This study suggests that impaired neurogenesis is an early critical event in the course of Alzheimer's disease that may underlie memory impairments, at least in part, and exacerbate neuronal vulnerability in the hippocampal formation and olfaction circuits. Furthermore, impaired neurogenesis is the result of both intrinsic pathology in neural progenitor cells and extrinsic neuropathology in the neurogenic niches. Finally, hyperphosphorylation of the microtubule-associated protein tau, a critical player in cell proliferation, neuronal maturation, and axonal transport, is a major contributor to impaired neurogenesis in Alzheimer's disease.

Inhibitory Effects of Phylligenin on the Proliferation of Cultured Rat Neural Progenitor Cells

Abstract － Neural progenitor cells (NPCs) differentiate into astrocytes, neurons and oligodendrocytes, which is controlled by various factors in brain. Recent evidences suggest that small molecules modulating the proliferation and differentiation of NPCs may have therapeutic value as well as the potential use as chemical probes. Phylligenin is a lignan with anti-inflammatory activity that is isolated from the fruits of Forsythia koreana. We investigated effects of phylligenin on proliferation and differentiation of NPCs. Treatment of phylligenin decreased the number of proliferating NPCs in culture without effects on the differentiation and survival of neural cells such as neurons and astrocytes. To examine the mechanism of the decreased NPCs number, we performed cell cycle analysis. Proliferation of NPCs was decreased via G1-S transition block by phylligenin treatment, and it was mediated by the increase of p21 level. However, phylligenin did not induce apoptosis of NPCs as determined by TUNEL assay and PARP cleavage. We also found that viability of glioma cell lines such as C6 and U87MG glioma cells, but not that of primary neuron and astrocyte, was inhibited by phylligenin. These results suggest that phylligenin selectively inhibits proliferation of rapidly growing cells such as neural stem cells and glioma cells. Given that the possible role of brain tumor stem cells in the pathology of brain cancers, the inhibitory effects of phylligenin might be useful in the development of new therapeutic agents against brain cancers.Keywords: Neural progenitor cells, Phylligenin, Proliferation, p21, Glioma

Genetic instability and diminished differentiation capacity in long-term cultured mouse neurosphere cells

The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.

Fas activation increases neural progenitor cell survival

Although there is a sizable amount of research focusing on adult neural progenitor cells (NPCs) as a therapeutic approach for many neurodegenerative diseases, including multiple sclerosis, little is known about the pathways that govern NPC survival and apoptosis. Fas, a member of the death receptor superfamily, plays a well-characterized role in the immune system, but its function in neural stem cells remains uncertain. Our study focuses on the effects of Fas on NPC survival in vitro. Activation of Fas by recombinant Fas ligand (FasL) did not induce apoptosis in murine NPCs in culture. In fact, both an increase in the amount of viable cells and a decrease in apoptotic and dying cells were observed with FasL treatment. Our data indicate that FasL-mediated adult NPC neuroprotection is characterized by a reduction in apoptosis, but not increased proliferation. Further investigation of this effect revealed that the antiapoptotic effects of FasL are mediated by the up-regulation of Birc3, an inhibitor of apoptosis protein (IAP). Conversely, the observed effect is not the result of altered caspase activation or FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein) up-regulation, which is known to inhibit caspase-8-mediated cell death in T cells. Our data indicate that murine adult NPCs are resistant to FasL-induced cell death. Activation of Fas increased cell survival by decreasing apoptosis through Birc3 up-regulation. These results describe a novel pathway involved in NPC survival.

Sonic Hedgehog Regulates Ischemia/Hypoxia-Induced Neural Progenitor Proliferation

Sonic hedgehog (Shh) protein is required for the maintenance of neural progenitor cells (NPCs) in the embryonic and adult hippocampus. Brain ischemia causes increased proliferation of hippocampal NPCs. We therefore examined whether Shh regulates the increase in proliferation of NPCs after ischemia/hypoxia. Male SV129 mice were exposed to a 20-minute middle cerebral artery occlusion; hippocampi were then analyzed for Shh mRNA and protein expression by real-time polymerase chain reaction, immunoblot, and immunohistochemistry. Primary cell cultures of neurons, astrocytes, and NPCs were exposed to 16 hours of hypoxia (1% O(2)) and analyzed by real-time polymerase chain reaction and immunoblot for Shh expression. Proliferation of NPCs, in vivo and in vitro, was measured by bromodeoxyuridine incorporation. Among the cell types examined in vitro, only NPC and neurons increased Shh mRNA under hypoxic conditions. Furthermore, hypoxia increased proliferation of NPCs and this proliferation was enhanced by the addition of recombinant Shh or blocked by the pathway-specific inhibitor, cyclopamine. Middle cerebral artery occlusion was associated with a transient 2-fold increase in the mRNA encoding both Shh and its transcription factor, Gli1, 0.5 days after ischemia. Within the hippocampus, Shh protein was increased approximately 3-fold 3 and 7 days after ischemia and was observed predominantly within cells in the CA3 and hilar regions. Shh was expressed only in mature neurons. In vivo, cyclopamine suppressed ischemia-induced proliferation of subgranular NPCs. The Shh pathway plays a role in the proliferation of NPCs induced by ischemia/hypoxia and might participate in injury remodeling.

Protection of neurons derived from human neural progenitor cells by veratridine

The survival of developing dopaminergic neurons has been shown to be modulated by voltage-dependent mechanisms. Manipulation of these mechanisms in human neural progenitor cell cultures could improve the survival of immature dopaminergic neurons, and therefore aid research into pharmacological and cell replacement therapies for Parkinson's disease. Here, we examined the effect of the Na+ channel agonist veratridine on the human fetal neural progenitor ReNcell VM cell line. Neuronal differentiation was determined by immunocytochemistry, whereas patch clamp recordings showed the expression of functional voltage-gated sodium channels. Our results show that veratridine is neuroprotective in human fetal neural progenitor cells, which may benefit studies investigating neuronal development by reducing premature death amongst developing neurons.

Mitochondrial Fragmentation Is Involved in Methamphetamine-Induced Cell Death in Rat Hippocampal Neural Progenitor Cells

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca2+) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.

Fourteen compounds and their derivatives for the treatment of diseases and injuries characterized by reduced neurogenesis and neurodegeneration

The application is in the field of adult neurogenesis and neuronal degeneration and regeneration. It aims to characterize the activity of 15 compounds and their derivatives on adult neurogenesis, apoptosis, necrosis and neuron dysfunction in vitro. The activity of the compounds and their derivatives was tested on cultures of adult human-derived neural progenitor and stem cells, in presence of staurosporine, peptide β-amyloid and hydrogen peroxide, and was assessed by immunocytology. The compounds and their derivatives stimulate the differentiation of adult-derived neural progenitor and stem cells into the neuronal pathway, and inhibit staurosporine-induced apoptosis, peptide β-amyloid-induced necrosis and hydrogen peroxide-induced neuron dysfunction. The application claims the use of the 15 compounds and their derivatives for the treatment of diseases and injuries particularly characterized by reduced neurogenesis and neuronal loss, including Alzheimer's disease (AD), depression, cerebral strokes and traumatic brain and spinal cord injuries.

Isoflurane Inhibits Growth but Does Not Cause Cell Death in Hippocampal Neural Precursor Cells Grown in Culture

Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells. Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers. Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure. The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.

High susceptibility of cortical neural progenitor cells to trimethyltin toxicity: Involvement of both caspases and calpain in cell death

Neural progenitor cells play an essential role in both the developing embryonic nervous system and in the adult brain, where the capacity for self-renewal would be important for normal brain functions. In the present study, we used embryonic cortical neural progenitor cells to investigate the effects of trimethyltin chloride (TMT) on the survival of neural progenitor cells. In cultures of cortical neural progenitor cells, the formation of round neurospheres was observed in the presence of epidermal growth factor and basic fibroblast growth factor within 9 days in vitro. The neurospheres were then harvested for subsequent replating and culturing for assessment of cell viability in either the presence or absence of TMT at the concentration of 5 μM. Lasting exposure to TMT produced not only nuclear condensation in the cells in a time-dependent manner over a period of 6–24 h, but also the release of lactate dehydrogenase into the culture medium. Immunoblot and immunocytochemical analyses revealed that TMT had the ability to activate both caspase-3 and calpain, as well as to cause nuclear translocation of deoxyribonuclease II, which is located within cytoplasm in intact cells. Additionally, treatment with a calpain inhibitor [ trans-epoxysuccinyl- l-leucylamido-(4-guanidino) butane] and a caspase inhibitor [Z-Val-Ala-Asp(OMe)-CH 2F] produced a significant reduction in damaged cells induced by TMT. Taken together, our data indicate that neural progenitor cells are highly susceptible to TMT in undergoing cell death via the activation of 2 parallel pathways, ones involving calpain and the other, caspase-3.

Lithium enhances the neuronal differentiation of neural progenitor cellsin vitroand after transplantation into the avulsed ventral horn of adult rats through the secretion of brain‐derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo. Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.

A Nuclear Magnetic Resonance Biomarker for Neural Progenitor Cells: Is It All Neurogenesis?

In vivo visualization of endogenous neural progenitor cells (NPCs) is crucial to advance stem cell research and will be essential to ensure the safety and efficacy of neurogenesis-based therapies. Magnetic resonance spectroscopic imaging (i.e., spatially resolved spectroscopy in vivo) is a highly promising technique by which to investigate endogenous neurogenesis noninvasively. A distinct feature in nuclear magnetic resonance spectra (i.e., a lipid signal at 1.28 ppm) was recently attributed specifically to NPCs in vitro and to neurogenic regions in vivo. Here, we demonstrate that although this 1.28-ppm biomarker is present in NPC cultures, it is not specific for the latter. The 1.28-ppm marker was also evident in mesenchymal stem cells and in non-stem cell lines. Moreover, it was absent in freshly isolated NPCs but appeared under conditions favoring growth arrest or apoptosis; it is initiated by induction of apoptosis and correlates with the appearance of mobile lipid droplets. Thus, although the 1.28-ppm signal cannot be considered as a specific biomarker for NPCs, it might still serve as a sensor for processes that are tightly associated with neurogenesis and NPCs in vivo, such as apoptosis or stem cell quiescence. However, this requires further experimental evidence. The present work clearly urges the identification of additional biomarkers for NPCs and for neurogenesis.

Effective Cryopreservation of Neural Stem or Progenitor Cells without Serum or Proteins by Vitrification.

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 μl sterile straw placed in 500 μl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Defective cell-cell adhesion in hyh mutant mice: a pathogenic mechanism underlying cerebral ventricular surface disruption and hydrocephalus?

Background The hyh mutant mice develop a severe, progressive hydrocephalus accompanied by disruption of the cerebral ventricular surface and distortion of the brain architecture. Although a point mutation (M105I) in alpha-SNAP protein has been identified as responsible of the hyh phenotype, many of the pathogenic mechanisms underlying this process remain unknown. Considering that alpha-SNAP is involved in the vesicular traffic of proteins to plasma membrane, we proposed that hyh mutation could result in a failure in cell-cell adhesion during brain development. To test this hypothesis, we decided to investigate (i) the presence of alpha-SNAP and N-cadherin in different regions of the brain, (ii) the ultrastructure of adherens junctions at ventricular surface, and (iii) the physiology of cell-cell adhesion in neural progenitor cells (NPCs) cultures.