Undifferentiated Neural Precursor Cells Research Articles

Toll-like receptors 2 and 4 differentially regulate the self-renewal and differentiation of spinal cord neural precursor cells

BackgroundToll-like receptors (TLRs) represent critical effectors in the host defense response against various pathogens; however, their known function during development has also highlighted a potential role in cell fate determination and neural differentiation. While glial cells and neural precursor cells (NPCs) of the spinal cord express both TLR2 and TLR4, their influence on self-renewal and cell differentiation remains incompletely described.MethodsTLR2, TLR4 knock-out and the wild type mice were employed for spinal cord tissue analysis and NPCs isolation at early post-natal stage. Sox2, FoxJ1 and Ki67 expression among others served to identify the undifferentiated and proliferative NPCs; GFAP, Olig2 and β-III-tubulin markers served to identify astrocytes, oligodendrocytes and neurons respectively after NPC spontaneous differentiation. Multiple comparisons were analyzed using one-way ANOVA, with appropriate corrections such as Tukey's post hoc tests used for comparisons.ResultsWe discovered that the deletion of TLR2 or TLR4 significantly reduced the number of Sox2-expressing NPCs in the neonatal mouse spinal cord. While TLR2-knockout NPCs displayed enhanced self-renewal, increased proliferation and apoptosis, and delayed neural differentiation, the absence of TLR4 promoted the neural differentiation of NPCs without affecting proliferation, producing long projecting neurons. TLR4 knock-out NPCs showed significantly higher expression of Neurogenin1, that would be involved in the activation of this neurogenic program by a ligand and microenvironment-independent mechanism. Interestingly, the absence of both TLR2 and TLR4, which induces also a significant reduction in the expression of TLR1, in NPCs impeded oligodendrocyte precursor cell maturation to a similar degree.ConclusionsOur data suggest that Toll-like receptors are needed to maintain Sox2 positive neural progenitors in the spinal cord, however possess distinct regulatory roles in mouse neonatal spinal cord NPCs—while TLR2 and TLR4 play a similar role in oligodendrocytic differentiation, they differentially influence neural differentiation.

Culturing Mouse Fetal Neural Precursor Cells in a Free-Floating Serum-Free Condition.

Neural precursor cells (NPCs) are a renewable cell source that can proliferate and expand for long periods of time and give rise to the main neural cell types of the central nervous system (CNS). Establishing simple and reproducible growth culture conditions is of great importance to study the biology of NPCs and to understand the molecular basis of their behavior in healthy and diseased conditions.Here, we describe a simple free-floating , serum-free culture condition, known as the neurosphere assay, which is the most commonly used method for the isolation and expansion of NPCs harvested from the adult and fetal CNS. This culture system will result in large numbers of undifferentiated NPC progenies that represents a useful cell source for many in vitro and in vivo applications.

Cystatin B-deficiency triggers ectopic histone H3 tail cleavage during neurogenesis

Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency.

Protective Role of Hypothermia Against Heat Stress in Differentiated and Undifferentiated Human Neural Precursor Cells: A Differential Approach for the Treatment of Traumatic Brain Injury

Introduction:The present study aimed to explore protective mechanisms of hypothermia against mild cold and heat stress on highly proliferative homogeneous human Neural Precursor Cells (NPCs) derived from Subventricular Zone (SVZ) of human fetal brain.Methods:CD133+ve enriched undifferentiated and differentiated human NPCs were exposed to heat stress at 42°C. Then, Western-blot quantification was performed using Hsp-70 (70 kilodalton heat shock proteins) recombinant protein. Finally, changes in pluripotency and Hsp-70 expression were measured using immunofluorescence staining and RT-qPCR (Quantitative reverse transcription PCR) analysis, respectively.Results:Heat stress resulted in abnormal neurospheres development. The apoptosis rate was enhanced during long-term in vitro culture of neurospheres. Neurogenic differentiation reduced and showed aberrent phenotypes during heat stress. After hypothermia treatment significant improvement in neurospheres and neuronal cell morphology was observed.Conclusion:Mild-hypothermia treatment induces attenuated heat shock response against heat stress resulting in induced HSP-70 expression that significantly improves structure and function of both undifferentiated human NPCs and differentiated neurons.

Human-induced pluripotent stem cells generated from intervertebral disc cells improve neurologic functions in spinal cord injury.

IntroductionInduced pluripotent stem cells (iPSCs) have emerged as a promising cell source for immune-compatible cell therapy. Although a variety of somatic cells have been tried for iPSC generation, it is still of great interest to test new cell types, especially those which are hardly obtainable in a normal situation.MethodsIn this study, we generated iPSCs by using the cells originated from intervertebral disc which were removed during a spinal operation after spinal cord injury. We investigated the pluripotency of disc cell-derived iPSCs (diPSCs) and neural differentiation capability as well as therapeutic effect in spinal cord injury.ResultsThe diPSCs displayed similar characteristics to human embryonic stem cells and were efficiently differentiated into neural precursor cells (NPCs) with the capability of differentiation into mature neurons in vitro.When the diPSC-derived NPCs were transplanted into mice 9 days after spinal cord injury, we detected a significant amelioration of hindlimb dysfunction during follow-up recovery periods. Histological analysis at 5 weeks after transplantation identified undifferentiated human NPCs (Nestin+) as well as early (Tuj1+) and mature (MAP2+) neurons derived from the transplanted NPCs. Furthermore, NPC transplantation demonstrated a preventive effect on spinal cord degeneration resulting from the secondary injury.ConclusionThis study revealed that intervertebral discs removed during surgery for spinal stabilization after spinal cord injury, previously considered a “waste” tissue, may provide a unique opportunity to study iPSCs derived from difficult-to-access somatic cells and a useful therapeutic resource for autologous cell replacement therapy in spinal cord injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0118-x) contains supplementary material, which is available to authorized users.

Biphasic monopolar electrical stimulation induces rapid and directed galvanotaxis in adult subependymal neural precursors.

IntroductionFollowing injury such as stroke, adult mammalian subependymal neural precursor cells (NPCs) are induced to proliferate and migrate toward the lesion site where they differentiate into neural cells, albeit with limited efficacy. We are interested in enhancing this migratory ability of NPCs with the long-term goal of promoting neural repair. Herein we build on our previous studies demonstrating that direct current electric fields (DCEFs) promote rapid and cathode-directed migration of undifferentiated adult NPCs (but not differentiated phenotypes) - a phenomenon known as galvanotaxis. While galvanotaxis represents a promising strategy to promote NPC recruitment to lesion sites, stimulation of neural tissue with DCEFs is not a clinically-viable strategy due to the associated accumulation of charge and toxic byproducts. Balanced biphasic waveforms prevent the accumulation of charge and thus are outside of the limitations of DCEFs. In this study, we investigated the effects of balanced biphasic electrical stimulation on the migratory behaviour of undifferentiated subependymal NPCs and their differentiated progeny.MethodsNPCs were isolated from the subependymal zone of adult mouse brains and cultured in a NPC colony-forming assay to form neurospheres. Neurospheres were plated onto galvanotaxis chambers in conditions that either promoted maintenance in an undifferentiated state or promoted differentiation into mature phenotypes. Chambers containing cells were then time-lapse imaged in the presence of either biphasic monopolar, or biphasic bipolar electrical stimulation, or in the complete absence of electrical stimulation. Single cell migration was subsequently tracked and the cells’ magnitude of velocity, directedness and tortuosity were quantified.ResultsWe demonstrate, for the first time, the use of balanced biphasic electric fields to induce galvanotaxis of NPCs. Undifferentiated adult mouse subependymal NPCs exposed to biphasic monopolar stimulation undergo rapid and directed migration toward the cathode. In contrast, both biphasic bipolar stimulation and the lack of electrical stimulation produced non-directed migration of NPCs. Notably, NPCs induced to differentiate into mature phenotypes prior to exposure to electrical stimulation do not migrate in the presence or absence of biphasic stimulation.ConclusionWe purport that balanced biphasic stimulation represents a clinically-viable technique for mobilizing NPCs that may be integrated into strategies for promoting endogenous neurorepair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0049-6) contains supplementary material, which is available to authorized users.

Tcf3 represses Wnt-β-catenin signaling and maintains neural stem cell population during neocortical development.

During mouse neocortical development, the Wnt–β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs). Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintenance of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3 (also known as Tcf7l1) contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs) committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene Neurogenin1 (Neurog1) and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1 (also known as Tcf7) and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation.

SC1/PRDM4 recruits PRMT5 to control the timing of neural precursor differentiation in developing neural stem cells

AbstractDuring cortical development, neural stem cells (NSCs) switch from proliferative to neuron-generating asymmetric divisions. Here we investigated the role of Schwann cell factor 1 (SC1/PRDM4), a transcriptional repressor highly expressed in the developing nervous system, during NSCs development. We found that SC1 protein levels were down-regulated in newly differentiating neurons, while remaining high in undifferentiated NSCs, suggesting an asymmetric inheritance of SC1. Knockdown of SC1 in the NSCs led to precocious differentiation of neurons and its overexpression led to an increase in Nestin-expressing precursors. We found that SC1, through its amino-terminus, recruits the chromatin modifier PRMT5, a histone arginine methyltransferase that catalyses histone H4R3 symmetric dimethylation (H4R3me2s). Mutations disrupting SC1/PRMT5 interaction resulted in loss of SC1-mediated increase in undifferentiated neural precursor cells. Our data demonstrate that SC1 and PRMT5 are components of an epigenetic regulatory complex that provides an epigenetic signature of a “stem-like” cellular state in the NSCs and which may be asymmetrically inherited during neurogenic divisions.

SC1/PRDM4 recruits PRMT5 to control the timing of neural precursor differentiation in developing neural stem cells

During cortical development, neural stem cells (NSCs) switch from proliferative to neuron-generating asymmetric divisions. Here we investigated the role of Schwann cell factor 1 (SC1/PRDM4), a transcriptional repressor highly expressed in the developing nervous system, during NSCs development. We found that SC1 protein levels were down-regulated in newly differentiating neurons, while remaining high in undifferentiated NSCs, suggesting an asymmetric inheritance of SC1. Knockdown of SC1 in the NSCs led to precocious differentiation of neurons and its overexpression led to an increase in Nestin-expressing precursors. We found that SC1, through its amino-terminus, recruits the chromatin modifier PRMT5, a histone arginine methyltransferase that catalyses histone H4R3 symmetric dimethylation (H4R3me2s). Mutations disrupting SC1/PRMT5 interaction resulted in loss of SC1-mediated increase in undifferentiated neural precursor cells. Our data demonstrate that SC1 and PRMT5 are components of an epigenetic regulatory complex that provides an epigenetic signature of a “stem-like” cellular state in the NSCs and which may be asymmetrically inherited during neurogenic divisions.

Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and synaptogenesis

Elevated levels of serum unconjugated bilirubin (UCB) in the first weeks of life may lead to long-term neurologic impairment. We previously reported that an early exposure of developing neurons to UCB, in conditions mimicking moderate to severe neonatal jaundice, leads to neuritic atrophy and cell death. Here, we have further analyzed the effect of UCB on nerve cell differentiation and neuronal development, addressing how UCB may affect the viability of undifferentiated neural precursor cells and their fate decisions, as well as the development of hippocampal neurons in terms of dendritic and axonal elongation and branching, the axonal growth cone morphology, and the establishment of dendritic spines and synapses. Our results indicate that UCB reduces the viability of proliferating neural precursors, decreases neurogenesis without affecting astrogliogenesis, and increases cellular dysfunction in differentiating cells. In addition, an early exposure of neurons to UCB decreases the number of dendritic and axonal branches at 3 and 9 days in vitro (DIV), and a higher number of neurons showed a smaller growth cone area. UCB-treated neurons also reveal a decreased density of dendritic spines and synapses at 21 DIV. Such deleterious role of UCB in neuronal differentiation, development, and plasticity may compromise the performance of the brain in later life.

Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation

Neural precursor cells (NPCs) differentiate into neurons, astrocytes, and oligodendrocytes in response to intrinsic and extrinsic changes. Notch signals maintain undifferentiated NPCs, but the mechanisms underlying the neuronal differentiation are largely unknown. We show that SIRT1, an NAD(+)-dependent histone deacetylase, modulates neuronal differentiation. SIRT1 was found in the cytoplasm of embryonic and adult NPCs and was transiently localized in the nucleus in response to differentiation stimulus. SIRT1 started to translocate into the nucleus within 10 min after the transfer of NPCs into differentiation conditions, stayed in the nucleus, and then gradually retranslocated to the cytoplasm after several hours. The number of neurospheres that generated Tuj1(+) neurons was significantly decreased by pharmacological inhibitors of SIRT1, dominant-negative SIRT1 and SIRT1-siRNA, whereas overexpression of SIRT1, but not that of cytoplasm-localized mutant SIRT1, enhanced neuronal differentiation and decreased Hes1 expression. Expression of SIRT1-siRNA impaired neuronal differentiation and migration of NPCs into the cortical plate in the embryonic brain. Nuclear receptor corepressor (N-CoR), which has been reported to bind SIRT1, promoted neuronal differentiation and synergistically increased the number of Tuj1(+) neurons with SIRT1, and both bound the Hes1 promoter region in differentiating NPCs. Hes1 transactivation by Notch1 was inhibited by SIRT1 and/or N-CoR. Our study indicated that SIRT1 is a player of repressing Notch1-Hes1 signaling pathway, and its transient translocation into the nucleus may have a role in the differentiation of NPCs.

Neural precursor cell influences on blood–brain barrier characteristics in rat brain endothelial cells

This study explores the effects of neural precursor cells (NPCs) on barrier characteristics in brain vasculature. Primary rat brain endothelial cells were exposed to conditioned medium from NPCs isolated from day 14 embryonic rat brains and maintained as free-floating undifferentiated neurospheres. Such exposure increased brain endothelial transcript levels of the mdr1a but not mdr1b gene encoding P-glycoprotein (Pgp) and reduced proliferation but did not alter transendothelial resistance (TER). These effects were compared to those seen following co-culture with differentiating NPCs or with primary astrocytes. NPCs, if grown adherent, differentiate into glial and neuronal cells as assessed by immunocytochemical and mRNA analysis. Brain endothelial cells when co-cultured with these cells also showed reduced proliferation and enhanced mdr1a expression, but in addition increased TER. Similar increases were observed in co-culture with astrocytes. These results suggest that undifferentiated NPCs produce factors that influence Pgp expression whereas their progeny also affect tight junction integrity.

Neural precursor cell susceptibility to human cytomegalovirus diverges along glial or neuronal differentiation pathways

Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain-derived NPCs (nestin(+), A2B5(+), CD133(+)) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP(+), nestin(-), A2B5(-)), viral expression was retained. However, differentiation of NPCs in medium containing platelet-derived growth factor and brain-derived neurotropic factor, conditions that support the development of neurons (Tuj-1(+), nestin(-), A2B5(-)), resulted in reduced viral expression, with corresponding decreased CMV major immediate-early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)-beta isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.

Comparison of neural precursor cell fate in second trimester human brain and spinal cord

Neural transplantation holds promise for the treatment of traumatic brain and spinal cord injury by replacing lost cellular elements as well as repairing neural damage. Fetal human stem cells derived from central nervous system (CNS) tissue are potential transplantable sources for all cell types found in the mature human nervous system including neurons, astrocytes and oligodendroglia. Although nearly all areas of the fetal human neuraxis contain undifferentiated neural precursor cells, the phenotypic fate of the daughter cells might vary from one region to another during a specific developmental period. The purpose of this study was to compare the various cell types derived from neural precursors cultured from second trimester fetal human brain and spinal cord. To this end, brains (n = 8) and spinal cords (n = 8) of 15-24 week fetuses were dissociated and grown in culture medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (FGF) and leukemia inhibitory factor (LIF). The proliferating precursor cells from both brain and spinal cord grew as spherical masses that were plated on laminin-coated dishes after seven days in culture. During the next 5-7 days, the cells that emerged from these spheres were fixed and processed for immunocytochemistry. Brain derived spheres gave rise to cells expressing antigens specific for neurons (MAP-2ab and neuron specific-intermediate filaments), astrocytes (GFAP) and oligodendrocytes (A007). In contrast, cells that emerged from spinal cord derived spheres were only immunoreactive for GFAP. These data suggest that neuroepithelial precursor cells from different CNS regions, although similar in their responsiveness to proliferative growth factors, might differ in their ability to generate different cell types in the adult CNS. [Neurol Res 2001; 23: 260-266]

Neurotransplantation in neurodegenerative disease: a survey of relevant issues in developmental neurobiology.

Neural development and transplantation therapies in neurodegenerative disease share a particular feature. In both cases, undifferentiated neural precursor cells are required to differentiate into a range of neural cell types in a tissue-specific fashion. This similarity opens the possibility that the mechanisms that drive neural development play a similar role in CNS repair. In this chapter, two aspects of neural development are considered in terms of their relevance to CNS repair: the diversity of neural precursor cells and positional specification. We present evidence to suggest that neural stem cells have a degree of diversity that is beyond what might have been expected a priori. We also show that neural stem cells express genes that might encode a positional specification for these cells, and consider a number of hypotheses about the role of positional specification in CNS repair.