Monoamine oxidase A regulates neural differentiation of murine embryonic stem cells

Monoamine oxidase (MAO) A is the major metabolizing enzyme of serotonin (5-hydroxytryptamine, 5-HT) which regulates early brain development. In this study, wild-type (WT) and MAO A^neo embryonic stem (ES) cell lines were established from the inner cell mass of murine blastocysts and their characteristics during ES and differentiating stages were studied. Our results show that the differentiation to neural cells in MAO A^neo ES cells was reduced compared to WT, suggesting MAO A played a regulatory role in stem cells neural differentiation.     

Psychiatric symptoms and CAG repeats in neurologically asymptomatic Huntington's disease gene carriers.

The putative relationship between the psychiatric profile of a sample of neurologically asymptomatic Huntington's disease gene carriers and CAG repeats was investigated. The psychiatric assessments (by consultant psychiatrist and computerised battery) were undertaken before the genetic testing was carried out. In this way, the informational distortions caused by neurological and cognitive deficits were avoided. The hypothesis that there is a relationship between psychiatric and CAG repeats was tested by seeking direct correlations between psychiatric systems and CAG repeats, and also by correcting the correlation by the number of years above or below the estimated age of onset in Huntington's disease. Scores for irritability and cognitive failures were high in the sample. There was no correlation between any psychiatric variable and CAG repeats. Possible explanations for this lack of correlations are discussed.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

Induced neuronal differentiation of human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells capable of forming differentiated embryoid bodies (EBs) in culture. We examined the ability of growth factors under controlled conditions to increase the number of human ES cell-derived neurons. Retinoic acid (RA) and nerve growth factor (betaNGF) were found to be potent enhancers of neuronal differentiation, eliciting extensive outgrowth of processes and the expression of neuron-specific molecules. Our findings show that human ES cells have great potential to become an unlimited cell source for neurons in culture. These cells may then be used in transplantation therapies for neural pathologies.     

Folic acid deficiency inhibits neural rosette formation and neuronal differentiation from rhesus monkey embryonic stem cells

Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.     

Cell cycle exit and neuronal differentiation 1-engineered embryonic neural stem cells enhance neuronal differentiation and neurobehavioral recovery after experimental traumatic brain injury

Our previous study showed that cell cycle exit and neuronal differentiation 1(CEND1)may participate in neural stem cell cycle exit and oriented differentiation.However,whether CEND1-transfected neural stem cells can improve the prognosis of traumatic brain injury remained unclear.In this study,we performed quantitative proteomic analysis and found that after traumatic brain injury,CEND1 expression was downregulated in mouse brain tissue.Three days after traumatic brain injury,we transplanted CEND1-transfected neural stem cells into the area surrounding the injury site.We found that at 5 weeks after traumatic brain injury,transplantation of CEND1-transfected neural stem cells markedly alleviated brain atrophy and greatly improved neurological function.In vivo and in vitro results indicate that CEND1 overexpression inhibited the proliferation of neural stem cells,but significantly promoted their neuronal differentiation.Additionally,CEND1 overexpression reduced protein levels of Notch1 and cyclin D1,but increased levels of p21 in CEND1-transfected neural stem cells.Treatment with CEND1-transfected neural stem cells was superior to similar treatment without CEND1 transfection.These findings suggest that transplantation of CEND1-transfected neural stem cells is a promising cell therapy for traumatic brain injury.This study was approved by the Animal Ethics Committee of the School of Biomedical Engineering of Shanghai Jiao Tong University,China(approval No.2016034)on November 25,2016.     

Matrigel supports survival and neuronal differentiation of grafted embryonic stem cell‐derived neural precursor cells

Cell replacement therapy holds great promise as a means of treating neurological disorders, including Parkinson's disease. However, one of the major obstacles to the success of this treatment is the low survival rate of grafted cells, which probably results from mechanical damage, acute inflammation, and immunological rejection. To overcome this problem, we investigated the effect of different types of extracellular matrix (ECM) on the survival and differentiation of embryonic stem (ES) cell‐derived neural precursor cells (NPCs). We tested materials from natural sources, including collagen, ornithine/laminin, and growth factor‐reduced Matrigel (gfrMG), as well as the synthetic biomaterial PuraMatrix, which consists of self‐assembling polypeptides. GfrMG efficiently supported cell survival, migration, and neurite outgrowth in vitro and promoted proliferation of grafted cells in vivo, resulting in larger graft volume and an increase in the number of TH‐positive dopaminergic neurons in the graft. GfrMG did not induce dopaminergic differentiation directly; rather, it reduced the invasion of pan‐leukocytic CD45‐positive cells into the graft. Insofar as the inflammatory or immune response in the host brain inhibits neuronal differentiation of grafted NPCs, gfrMG may increase the number of TH‐positive cells by suppressing this effect. Thus, gfrMG appears to provide a suitable scaffold that supports survival and differentiation of NPCs. However, because it is derived from mouse sarcomas, a human‐derived matrix or synthetic biomaterial must be developed for clinical applications. © 2009 Wiley‐Liss, Inc.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

Interferon-gamma but not TNF alpha promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells

Neural trauma, such as traumatic brain injury or stroke, results in a vigorous inflammatory response at and near the site of injury, with cytokine production by endogenous glial cells and invading immune cells. Little is known of the effect that these cytokines have on neural stem cell function. Here we examine the effects of two inflammatory cytokines, interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNFalpha), on adult neural stem cells. Neural stem cells grown in the presence of either cytokine failed to generate neurospheres. Cytotoxicity assays showed that TNF alpha but not IFN gamma was toxic to the neural stem cells under proliferative conditions. Under differentiating conditions, neither cytokine was toxic; however, IFN gamma enhanced neuronal differentiation, rapidly increasing beta III-tubulin positive cell numbers 3-4 fold and inhibiting astrocyte generation. Furthermore, neurite outgrowth and the number of neurites per neuron was enhanced in cells differentiated in the presence of IFN gamma. Therefore, both inflammatory cytokines examined have substantial, but different effects on neural stem cell function and suggests that regulation of the inflammatory environment following brain injury may influence the ability of neural stem cells to repair the damage.     

Astrocytogenesis of embryonic stem-cell-derived neural stem cells: Default differentiation

Neural stem cells differentiate from embryonic stem cells via formation of neural stem spheres under free-floating conditions in astrocyte-conditioned medium. Subsequent culture of neural stem spheres on an adhesive substrate with fibroblast growth factor-2 promotes the migration of neural stem cells onto the substrate, resulting in an increase in the number of cells. These embryonic stem cell-derived neural stem cells can be differentiated almost exclusively into astrocytes by withdrawing fibroblast growth factor-2 from the medium without any additional instructions.     

Ascorbic acid responsive genes during neuronal differentiation of embryonic stem cells

Ascorbic acid has been reported to enhance differentiation of embryonic stem (ES) cells into neurons, however, the specific functions of ascorbic acid have not been defined yet. To address this issue, gene expression profiling was performed using cDNA microarray. Ascorbic acid increased the expressions of genes involved in neurogenesis, maturation, and neurotransmission. Furthermore, statistical analysis using Fisher's exact test revealed ascorbic acid significantly modulated the genes involved in cell adhesion and development category. These results provide information on the role for ascorbic acid during neuronal differentiation of ES cells and might contribute to large-scale generation of neurons for future clinical treatment.     

Clinical picture of patients with Huntington's disease in relation to the number of trinucleotide CAG repeats in IT-15 gene

Huntington's disease (HD) is a neurological condition of progressive course that results from abnormally increased number of CAG repeats within IT-15 gene, coding for huntington. The main symptoms consist of choraetic movements, dementia, and characteropathy. The aim of the present study was to search for possible correlation between the age of the onset of HD, time from the onset, clinical status of the patients, and CAG repeats number. Ten patients were studied altogether. Modified UHDRS (MUHDRS) was applied for the estimation of patients' clinical status. The number of CAG repeats in examination of the IT-15 gene was determined by polymerase chain reaction (PCR) and separation of radioisotope labelled PCR product against DNA size marker in polyacrylamide gel. A negative significant correlation was found between the CAG repeats number and the disease onset age (r = -0.67; p < 0.05) and MUHDRS score (r = 0.75; p < 0.05), as well. Negative significant correlation between time from the onset and MUHDRS score (r = -0.95; p < 0.05) and negative correlation between summarised: time from the onset and CAG number on the one site and MUHDRS on the other (p = -0.91) were found, as well. Our findings indicate an interdependence between CAG repeats number within the IT-15 gene, the course of the disease and the clinical status of HD patients.     

VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G-protein-coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB-derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT-PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB-derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.     

Fluoxetine promotes gliogenesis during neural differentiation in mouse embryonic stem cells

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed for treatment of mood disorders and depression, even during pregnancy and lactation. SSRIs are thought to be much safer than tricyclic antidepressants, with a low risk of embryonic toxicity. Several recent studies, however, have reported that fetal exposure to SSRIs increases the risk of adverse effects during fetal and neonatal development. This is consistent with our previous finding that fluoxetine, a prototypical SSRI, profoundly affected the viability of cultured embryonic stem (ES) cells as well as their ability to differentiate into cardiomyocytes. Furthermore, we found that fluoxetine induced fluctuations in ectodermal marker gene expression during ES cell differentiation, which suggests that fluoxetine may affect neural development. In the present study, we investigated the effects of fluoxetine on the process of differentiation from ES cells into neural cells using the stromal cell‐derived inducing activity (SDIA) method. Fluoxetine treatment was found to enhance the expression of glial marker genes following neural differentiation, as observed by immunocytochemical analysis or quantitative RT‐PCR. The promoter activity of glial marker genes was also significantly enhanced when cells were treated with fluoxetine, as observed by luciferase reporter assay. The expression of neuronal markers during ES cell differentiation into neural cells, on the other hand, was inhibited by fluoxetine treatment. In addition, FACS analysis revealed an increased population of glial cells in the differentiating ES cells treated with fluoxetine. These results suggest that fluoxetine could facilitate the differentiation of mouse ES cells into glial cell lineage, which may affect fetal neural development. © 2010 Wiley‐Liss, Inc.     

Staurosporine is a potent activator of neuronal,glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells

Staurosporine (STS), a broad spectrum protein kinase inhibitor, was previously shown to induce neurite outgrowth in several neuroblastoma cell lines. However, data on the neurotrophic potential of this alkaloid in embryonic stem cell systems were not available. Therefore, three mouse ES cell lines, IB10, RW4, and Bruce 4, were induced to enter neurogenesis in culture at low concentrations of STS. These cells differentiated into epidermal growth factor-responsive neural precursor cells, formed neurospheres, and further developed to neurons and astrocytes. The clonally derived neurospheres consisted of multipotent cells which exhibited some of the classical characteristics of early CNS stem cells and could be propagated in vitro. STS was antagonistic in several ways to retinoic acid (RA), a vitamin A metabolite, which promotes neuritogenesis. Results from RT-PCR experiments and inhibition studies with RA provided evidence that staurosporine exerted its neurotrophic effects through the induction of very late levels of the nerve growth factor and protein kinase C neurogenesis pathways.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopamincrgic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abeam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R＆D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/Fl2, 1% N2 supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differentiate in the DMEM/Fl2 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N2 additive.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells*☆

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tu     

Analysis of the expression and function of BRINP family genes during neuronal differentiation in mouse embryonic stem cell‐derived neural stem cells

We previously identified a novel family of genes, BRINP1, 2, and 3, that are predominantly and widely expressed in both the central nervous system (CNS) and peripheral nervous system (PNS). In the present study, we analyzed the expression pattern of three BRINP genes during differentiation of mouse embryonic stem (ES) cell‐derived neural stem cells (NSCs) and their effects on the cell‐cycle regulation of NSCs. While there was no significant expression of any BRINP‐mRNA expressed in mouse ES cells, BRINP 1 and 2‐mRNAs was expressed at high levels in the ES cell‐derived neural stem cells. Upon differentiation into neuronal cells in the presence of retinoic acid and BDNF, all three types of BRINP‐mRNA were induced with a similar time course peaking at day three of treatment. Upon differentiation into astroglial cells in the presence of serum, BRINP1‐mRNA was slightly up‐regulated, while BRINP2‐ and BRINP3‐mRNAs were almost abolished in the astrocytes. While 69.2, 26.1, and 7.7% of cells in a population of NSCs in the exponentially growing phase were in the G1, S and G2 phases, respectively, over‐expression of any one of the three BRINP genes completely abolished cells in the G2 phase and significantly reduced the cells in S phase to 11.8–13.8%. Based on these results, the physiological roles of induced BRINP genes in the cell‐cycle suppression of terminally differentiated post‐mitotic neurons are discussed. © 2009 Wiley‐Liss, Inc.