磷脂酶D1信号对神经干细胞向神经分化的重要影响

磷脂酶D1(PLD1)在细胞生长、存活、分化、膜转运和细胞骨架组织等多种功能的调控中发挥重要作用。近年来研究发现,PLD1在神经干细胞（NSCs）向神经元的分化中也起关键作用。PLD1参与多种信号通路如Rho家族GTP酶和Ca²⁺信号通路的调节,影响轴突生长、突触发育及其可塑性。因此,PLD1作为神经系统中一种重要的信号分子引起了广泛的关注。本文综述了PLD1的结构、功能、作用机制及其在NSCs向神经分化中的调控作用,对深入研究NSCs的分化和神经元的再生有重要的指导意义。     

Effects of sulforaphane on neural stem cell proliferation and differentiation

Sulforaphane (SFN) is a natural organosulfur compound with anti‐oxidant and anti‐inflammation properties. The objective of this study is to investigate the effect of SFN on the proliferation and differentiation of neural stem cells (NSC). NSCs were exposed to SFN at the concentrations ranging from 0.25 to 10 µM. Cell viability was evaluated with MTT assay and lactate dehydogenase (LDH) release assay. The proliferation of NSCs was evaluated with neurosphere formation assay and Ki‐67 staining. The level of Tuj‐1 was evaluated with immunostaining and Western blot to assess NSC neuronal differentiation. The expression of key proteins in the Wnt signaling pathway, including β‐catenin and cyclin D1, in response to SFN treatment or the Wnt inhibitor, DKK‐1, was determined by Western blotting. No significant cytotoxicity was seen for SFN on NSCs with SFN at concentrations of less than 10 µM. On the contrary, SFN of low concentrations stimulated cell proliferation and prominently increased neurosphere formation and NSC differentiation to neurons. SFN treatment upregulated Wnt signaling in the NSCs, whereas DKK‐1 attenuated the effects of SFN. SFN is a drug to promote NSC proliferation and neuronal differentiation when used at low concentrations. These protective effects are mediated by Wnt signaling pathway.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

ERK-mediated production of neurotrophic factors by astrocytes promotes neuronal stem cell differentiation by erythropoietin

Erythropoietin (EPO), a hematopoietic factor, is also required for normal brain development, and its receptor is localized in brain. Our previous study showed that EPO promotes differentiation of neuronal stem cells into astrocytes. Since astrocytes have influence on the neuronal function, we investigated whether EPO-activated astrocytes could stimulate differentiation of neuronal stem cells into neurons. EPO did not promote neuronal differentiation of neuronal stem cells isolated from 17 day embryos, however, neuronal differentiation was promoted when the neuronal stem cells were co-cultured with astrocyte isolated from post neonatal (Day 1) rat brain. Moreover, neuronal differentiation was further promoted when the neuronal stem cells were cultured with astrocyte culture medium treated by EPO (10U/ml) showing increase of morphological differentiation, and expression of neuronal differentiation marker proteins, neurofilament, and tyrosine hydroxylase. The promoting effect of EPO-treated astrocyte medium was also found in the differentiation of PC12 cells. EPO-promoted morphological differentiation of neuronal stem cells as well as astrocytes was dose dependently reduced by treatment with anti-EPO receptor antibodies in culture with astrocyte culture medium. To clarify whether EPO itself or via production of well-known neurotropic factor could promote neuronal cell differentiation, we determined the level of neurotropic factors in the EPO-treated astrocytes. Compared to untreated astrocytes, EPO-treated astrocytes increased about 2-fold in beta-NGF and 3-4-fold in BMP2, but did not increase BNDF and NT-3 levels. Since the previous study showed that extracellular signal-regulated kinase (ERK) is involved in activation of astrocytes by EPO, we determined whether generation of neurotrophic factor may also be involved with the ERK pathway. In the presence of ERK inhibitor, PD98059, the generation of beta-NGF was diminished in a dose dependent manner consistent with the inhibiting effect on neuronal differentiation. These data demonstrate that EPO promotes neuronal cell differentiation through increased release of beta-NGF and BMP2 from astrocytes, and this effect may be associated with ERK pathway signals.     

The neural stem cell niche

The neural stem cell niche defines a zone in which stem cells are retained after embryonic development for the production of new cells of the nervous system. This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, albeit one that is restricted to a few specific zones within the brain. Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions. Ultimately, based on the location of the niche, stem cells of the adult brain support regeneration in the dentate gyrus of the hippocampus and the olfactory bulb through neuron replacement. Here, we provide a summary of the current understanding of the organization and control mechanisms of the neural stem cell niche.     

Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation

Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC‐induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC‐differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo.     

Discovery of small molecules that enhance astrocyte differentiation in rat fetal neural stem cells

1,3,4-Oxadiazole derivatives were found to enhance astrocyte differentiation in rat fetal neural stem cells (NSCs). Differentiation activity was assessed by immunocytochemistry and analysis of mRNA expression of astrocyte markers, GFAP and S100. Compounds 7 and 8 showed approximately a two-fold increase in astrocyte differentiation without engagement of neuronal differentiation and detectable cytotoxicity.     

ES cell neural differentiation reveals a substantial number of novel ESTs

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease. Electronic Publication     

神经干细胞的分离、培养及应用前景

胚胎和成年哺乳动物脑内均存在神经干细胞,具有潜在的增值和分化能力。在一定条件下,神经干细胞可向多个方向分化,生成神经元和神经胶质细胞,这为利用神经干细胞进行中枢神经系统退行性病变和损伤的治疗打下基础。     

Alternating current electric field effects on neural stem cell viability and differentiation

Methods utilizing stem cells hold tremendous promise for tissue engineering applications; however, many issues must be worked out before these therapies can be routinely applied. Utilization of external cues for preimplantation expansion and differentiation offers a potentially viable approach to the use of stem cells in tissue engineering. The studies reported here focus on the response of murine neural stem cells encapsulated in alginate hydrogel beads to alternating current electric fields. Cell viability and differentiation was studied as a function of electric field magnitude and frequency. We applied fields of frequency (0.1–10) Hz, and found a marked peak in neural stem cell viability under oscillatory electric fields with a frequency of 1 Hz. We also found an enhanced propensity for astrocyte differentiation over neuronal differentiation in the 1 Hz cultures, as compared to the other field frequencies we studied. Published 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy

A considerable amount of retrospective data is available that describes putative mesenchymal stem cells (MSCs). However, there is still very little knowledge available that documents the properties of a MSC in its native environment. Although the precise identity of MSCs remains a challenge, further understanding of their biological properties will be greatly advanced by analyzing the mechanisms that govern their self-renewal and differentiation potential. This review begins with the current state of knowledge on the biology of MSCs, specifically with respect to their existence in the adult organism and postulation of their biological niche. While MSCs are considered suitable candidates for cell-based strategies owing to their intrinsic capacity to self-renew and differentiate, there is currently little information available regarding the molecular mechanisms that govern their stem cell potential. We propose here a model for the regulation of MSC differentiation, and recent findings regarding the regulation of MSC differentiation are discussed. Current research efforts focused on elucidating the mechanisms regulating MSC differentiation should facilitate the design of optimal in vitro culture conditions to enhance their clinical utility cell and gene therapy.     

beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system

beta-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development. We employed here two conditional beta-catenin mutant alleles to alter beta-catenin signaling in the central nervous system of mice: one allele to ablate beta-catenin and the second allele to express a constitutively active beta-catenin. The tissue mass of the spinal cord and brain is reduced after ablation of beta-catenin, and the neuronal precursor population is not maintained. In contrast, the spinal cord and brain of mice that express activated beta-catenin is much enlarged in mass, and the neuronal precursor population is increased in size. beta-Catenin signals are thus essential for the maintenance of proliferation of neuronal progenitors, controlling the size of the progenitor pool, and impinging on the decision of neuronal progenitors to proliferate or to differentiate.     

Cultured Rat Astrocytes Give Rise to Neural Stem Cells

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI), but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type 1 astrocytes taken from newborn rat cortex in which the subventricular zone and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.     

Signaling pathways of the early differentiation of neural stem cells by neurotrophin-3

Neurotrophin-3 (NT-3) is well known to play an important role in facilitating neuronal survival and differentiation during development. However, the mechanisms by which neurotrophin-3 promotes prolonged Akt/MAPK signaling at an early stage are not well understood. Here, we report that NT-3 works at an early stage of neuronal differentiation in mouse neural stem cells (NSCs). After treatment with NT-3 for 12h, more NSCs differentiated into neurons than did untreated cells. These findings demonstrated that stimulation with NT-3 causes NSCs to differentiate into neurons through a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the phosphorylated extracellular signal-regulated kinase (ERK) pathway. In addition, treatment with NT-3 induced neurite outgrowth by specific phosphorylation of p38 MAPK, which was accompanied by neuronal differentiation. Taken together, these results suggest that NT-3, along with the Trk C receptors in NSCs, might lead to the survival and neuronal differentiation of NSCs via two distinct downstream signaling pathways at an early stage of neuronal differentiation.