Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.     

Tenascin-R plays a role in neuroprotection via its distinct domains that coordinate to modulate the microglia function

Microglia are one of the main cell types activated by brain injury. In the present study, we have investigated how domains of the extracellular matrix molecule tenascin-R (TN-R) modulate microglia function. We found that epidermal growth factor-like repeats inhibited adhesion and migration of microglia via a protein kinase A-dependent mechanism. In contrast, fibronectin 6-8 repeats promoted adhesion and migration of the primary microglia via a protein kinase C-dependent mechanism. Both domains of TN-R induced an up-regulation in the secretion of cytokines, such as chemokine-induced cytokine 3 and tumor neurosis factor alpha. Interestingly, epidermal growth factor-like repeats and fibronectin 6-8 induced a dramatic up-regulation in the secretion of brain-derived neurotrophic factor/transforming growth factor-beta and nerve growth factor/transforming growth factor-beta, respectively, and conditioned medium from activated microglia was able to promote neurite outgrowth of N1E-115 cells and primary cortical neurons. These results suggest that TN-R plays a role in neuroprotection through distinct domains coordinating to modulate microglia function.     

Excitation-neurogenesis coupling in adult neural stem/progenitor cells

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. Here we demonstrate that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca(v)1.2/1.3 (L-type) Ca(2+) channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an "excitation-neurogenesis coupling rule" within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories.     

成体神经干(前体)细胞在中枢神经系统退行性病变中的修复作用

尽管传统概念长期认为成体哺乳动物中枢神经系统缺乏再生增殖能力,但近年来发现,在成体若干脑区内确实存在具有再生与分化能力的神经干或神经前体细胞。这些干细胞在正常倩况下仅表现较低的再生分化活动。不过,在神经退行性病变中,病灶区内的干细胞可被动员、激活,并以较高的速率分裂分化以及取代坏死的神经元或胶质细胞,达到自身原位修复的作用。许多神经生长和营养因子具有增强或抑制干细胞分裂秋或分化的能力,在神经退行性病变中,病灶区内外成熟或新生细胞即可通过表达这些因子,有效调节干细胞的活动和干细胞主导的修复过程。总之,成体神经干细胞可以积极参与急性或慢性神经组织损伤的修复,通过再生来提供新的神经元以及其他必需的细胞,以促进功能的恢复。     

How to keep proliferative neural stem/progenitor cells

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

Lhx8 promote differentiation of hippocampal neural stem/progenitor cells into cholinergic neurons in vitro

Lhx8, also named L3, is a recently identified member of the LIM homeobox gene family. Previously, we found acetylcholinesterase (AChE)-positive cells in fimbria?Cfornix (FF) transected rat hippocampal subgranular zone (SGZ). In the present study, we detected choline acetyltransferase (ChAT)-positive cholinergic cells in hippocampal SGZ after FF transaction, and these ChAT-positive cells were double labeled by Lhx8. Then we overexpressed Lhx8 during neural differentiation of hippocampal neural stem/progenitor cells on adherent conditions using lentivirus Lenti6.3-Lhx8. The result indicated that overexpression of Lhx8 did not affect the proportion of MAP2-positive neurons, but increased the proportion of ChAT-positive cells in vitro. These results suggested that FF-transected hippocampal niche promoted the ChAT/Lhx8-positive cholinergic neurons generation in rodent hippocampus, and Lhx8 was not associated with the MAP2-positive neurons differentiation on adherent conditions, but played a role in the specification of cholinergic neurons derived from hippocampal neural stem/progenitor cells in vitro.     

Stem cell-derived neural stem/progenitor cell supporting factor is an autocrine/paracrine survival factor for adult neural stem/progenitor cells

Recent evidence suggests that adult neural stem/progenitor cells (ANSCs) secrete autocrine/paracrine factors and that these intrinsic factors are involved in the maintenance of adult neurogenesis. We identified a novel secretory molecule, stem cell-derived neural stem/progenitor cell supporting factor (SDNSF), from adult hippocampal neural stem/progenitor cells by using the signal sequence trap method. The expression of SDNSF in adult central nervous system was localized to hippocampus including dentate gyrus, where the neurogenesis persists throughout life. In induced neurogenesis status seen in ischemically treated hippocampus, the expression of SDNSF was up-regulated. As functional aspects, SDNSF protein provided a dose-dependent survival effect for ANSC following basic fibroblast growth factor 2 (FGF-2) withdrawal. ANSCs treated by SDNSF also retain self-renewal potential and multipotency in the absence of FGF-2. However, SDNSF did not have mitogenic activity, nor was it a cofactor that promoted the mitogenic effects of FGF-2. These data suggested an important role of SDNSF as an autocrine/paracrine factor in maintaining stem cell potential and lifelong neurogenesis in adult central nervous system.     

Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.     

The effect of cannabichromene on adult neural stem/progenitor cells

Apart from the psychotropic compound Δ⁹-tetrahydrocannabinol (THC), evidence suggests that other non-psychotropic phytocannabinoids are also of potential clinical use. This study aimed at elucidating the effect of major non-THC phytocannabinoids on the fate of adult neural stem progenitor cells (NSPCs), which are an essential component of brain function in health as well as in pathology. We tested three compounds: cannabidiol, cannabigerol, and cannabichromene (CBC), and found that CBC has a positive effect on the viability of mouse NSPCs during differentiation in vitro. The expression of NSPC and astrocyte markers nestin and Glial fibrillary acidic protein (GFAP), respectively, was up- and down-regulated, respectively. CBC stimulated ERK1/2 phosphorylation; however, this effect had a slower onset in comparison to typical MAPK stimulation. A MEK inhibitor, U0126, antagonized the up-regulation of nestin but not the down-regulation of GFAP. Based on a previous report, we studied the potential involvement of the adenosine A1 receptor in the effect of CBC on these cells and found that the selective adenosine A1 receptor antagonist, DPCPX, counteracted both ERK1/2 phosphorylation and up-regulation of nestin by CBC, indicating that also adenosine is involved in these effects of CBC, but possibly not in CBC inhibitory effect on GFAP expression. Next, we measured ATP levels as an equilibrium marker of adenosine and found higher ATP levels during differentiation of NSPCs in the presence of CBC. Taken together, our results suggest that CBC raises the viability of NSPCs while inhibiting their differentiation into astroglia, possibly through up-regulation of ATP and adenosine signalling.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc.     

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment.     

Human neural stem/progenitor cells derived from embryonic stem cells and fetal nervous system present differences in immunogenicity and immunomodulatory potentials in vitro

To develop cell therapies for damaged nervous tissue with human neural stem/progenitor cells (hNPCs), the risk of an immune response and graft rejection must be considered. There are conflicting results and lack of knowledge concerning the immunocompetence of hNPCs of different origin. Here, we studied the immunogenicity and immunomodulatory potentials of hNPCs cultured under equivalent conditions after derivation from human embryonic stem cells (hESC-NPCs) or human fetal spinal cord tissue (hfNPCs). The expression patterns of human leukocyte antigen, co-stimulatory and adhesion molecules in hESC-NPCs and hfNPCs were relatively similar and mostly not affected by inflammatory cytokines. Unstimulated hfNPCs secreted more transforming growth factor-β1 (TGF-β1) and β2 but similar level of interleukin (IL)-10 compared to hESC-NPCs. In contrast to hfNPCs, hESC-NPCs displayed 4–6 fold increases in TGF-β1, TGF-β2 and IL-10 under inflammatory conditions. Both hNPCs reduced the alloreaction between allogeneic peripheral blood mononuclear cells (PBMCs) and up-regulated CD4⁺CD25⁺forkhead box P3 (FOXP3)⁺ T cells. However, hESC-NPCs but not hfNPCs dose-dependently triggered PBMC proliferation, which at least partly may be due to TGF-β signaling. To conclude, hESC-NPCs and hfNPCs displayed similarities but also significant differences in their immunocompetence and interaction with allogeneic PBMCs, differences may be crucial for the outcome of cell therapy.     

Accumulation of mutations and somatic selection in aging neural stem/progenitor cells

Genomic instability within somatic stem cells may lead to the accumulation of mutations and contribute to cancer or other age-related phenotypes. However, determining the frequency of mutations that differ among individual stem cells is difficult from whole tissue samples because each event is diluted in the total population of both stem cells and differentiated tissue. Here the ability to expand neural stem/progenitor cells clonally permitted measurement of genomic alterations derived from a single initial cell. C57Bl/6 x DBA/2 hybrid mice were used and PCR analysis with strain-specific primers was performed to detect loss of heterozygosity on nine different chromosomes for each neurosphere. The frequency with which changes occurred in neurospheres derived from 2-month- and 2-year-old mice was compared. In 15 neurospheres derived from young animals both parental chromosomes were present for all nine chromosome pairs. In contrast, 16/17 neurospheres from old animals demonstrated loss of heterozygosity (LOH) on one or more chromosomes and seven exhibited a complete deletion of at least one chromosomal region. For chromosomes 9 and 19 there is a significant bias in the allele that is lost where in each case the C57Bl/6 allele is retained in 6/6 neurospheres exhibiting LOH. These data suggest that aging leads to a substantial mutational load within the neural stem cell compartment which can be expected to affect the normal function of these cells. Furthermore, the retention of specific alleles for chromosomes 9 and 19 suggests that a subset of mutational events lead to an allele-specific survival advantage within the neural stem cell compartment.     

Direct stimulation of adult neural stem/progenitor cells in vitro and neurogenesis in vivo by salvianolic acid B

Background

Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates.

Methodology and Principal Findings

We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats.

Significance

Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

F-Spondin, expressed in somite regions avoided by neural crest cells, mediates inhibition of distinct somite domains to neural crest migration

Neural crest (NC) cells migrate exclusively into the rostral half of each sclerotome, where they avoid the dermomyotome and the paranotochordal sclerotome. F-spondin is expressed in these inhibitory regions and throughout the caudal halves. In vitro bioassays of NC spreading on substrates of rostral or caudal epithelial-half somites (RS or CS, respectively) revealed that NC cells adopt on RS a fibroblastic morphology, whereas on CS they fail to flatten. F-spondin inhibited flattening of NC cells on RS. Conversely, F-spondin antibodies prevented rounding up of NC cells on CS. Addition of F-spondin to trunk explants inhibited NC migration into the sclerotome, and treatment of embryos with anti-F-spondin antibodies yielded migration into otherwise inhibitory sites. Thus, somite-derived F-spondin is an inhibitory signal involved in patterning the segmental migration of NC cells and their topographical segregation within the RS.     

Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells

We investigated the localization and functional expression of the ABC transporter ABCB1 in human fetal neural stem/progenitor cells (hNSPCs). RT-PCR analysis revealed ABCB1 gene expression in hNSPCs. We found a single band in immunoblotted hNSPCs lysates probed with ABCB1 antibody, and detected ABCB1 at the hNSPCs cell membrane by immunocytochemistry and subcellular fractionation. ABCB1 inhibitors and substrate, and ATP-depleting agents enhanced hNSPCs' rhodamine 123 accumulation, and hNSPCs microsomes had vanadate-sensitive ATPase activity. ABCB1 and nestin expression decreased during hNSPCs differentiation, while the astroglial marker GFAP increased. ABCB1 may maintain hNSPCs in an undifferentiated state and could be a neural stem/progenitor marker.     

Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.