Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.     

新生大鼠海马源性神经干细胞的分离培养及其向胆碱能神经元定向诱导分化研究(英文)

本研究目的是从新生SD大鼠海马分离、培养神经干细胞并诱导其向胆碱能神经元方向分化。利用含b FGF(20ng/ml)和B27的无血清DMEM/F12培养基培养新生SD大鼠海马分离的具有自我更新和多向分化能力的细胞群,用免疫细胞化学技术检测巢蛋白(nestin),并于分化后分别检查特异性成熟神经细胞、星形胶质细胞、少突胶质细胞的标记抗原β微管蛋白(Tuj1 )、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷脂(Galc)的表达;用鸡胚骨骼肌提取液,诱导神经干细胞向胆碱能神经元方向分化。结果显示:从海马分离的细胞群具有自我更新能力,表达nestin,分化成熟后的细胞表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原;与对照组3. 9%相比,鸡胚骨骼肌提取液可以诱导这些细胞中的9. 6%分化成为胆碱能神经元。提示分离的细胞具有自我更新能力和多向分化潜能,是中枢神经系统的干细胞;在加有鸡胚骨骼肌提取液的培养基诱导下,能向胆碱能神经元方向分化。     

Multipotent and restricted precursors in the central nervous system

Acquisition of cell type‐specific properties in the nervous system is likely a process of sequential restriction in developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and EGF‐dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the generation of intermediate lineage‐restricted precursors that differ from each other and from multipotent stem cells. Neuronal precursors termed neuronal‐restricted precursors (NRPs), multiple classes of glial precursors termed glial‐restricted precursors (GRPs), oligodendrocyte‐type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non‐fetal source of such cells. Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat Rec (New Anat): 257:137–143, 1999. © 1999 Wiley‐Liss, Inc.     

Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium–valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications.     

Neural stem cells and regulation of cell number

Normal CNS development involves the sequential differentiation of multipotent stem cells. Alteration of the numbers of stem cells, their self-renewal ability, or their proliferative capacity will have major effects on the appropriate development of the nervous system. In this review, we discuss different mechanisms that regulate neural stem cell differentiation. Proliferation signals and cell cycle regulators may regulate cell kinetics or total number of cell divisions. Loss of trophic support and cytokine receptor activation may differentially contribute to the induction of cell death at specific stages of development. Signaling from differentiated progeny or asymmetric distribution of specific molecules may alter the self-renewal characteristics of stem cells. We conclude that the final decision of a cell to self-renew, differentiate or remain quiescent is dependent on an integration of multiple signaling pathways and at each instant will depend on cell density, metabolic state, ligand availability, type and levels of receptor expression, and downstream cross-talk between distinct signaling pathways.     

Multipotent and restricted precursors in the central nervous system.

Acquisition of cell type-specific properties in the nervous system is likely a process of sequential restriction in developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and EGF-dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the generation of intermediate lineage-restricted precursors that differ from each other and from multipotent stem cells. Neuronal precursors termed neuronal-restricted precursors (NRPs), multiple classes of glial precursors termed glial-restricted precursors (GRPs), oligodendrocyte-type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non-fetal source of such cells. Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat Rec (New Anat): 257:137-143, 1999.     

Neural stem-like cell line derived from a nonhematopoietic population of human umbilical cord blood

The ability of stem and progenitor cells to proliferate and differentiate into other lineages is widely viewed as a characteristic of stem cells. Previously, we have reported that cells from a CD34(-) (nonhematopoietic) adherent subpopulation of human cord blood can acquire a feature of multipotential neural progenitors in vitro. In the present study, using these cord blood-derived stem cells, we have established a clonal cell line termed HUCB-NSCs (human umbilical cord blood-neural stem cells) that expresses several neural antigens and has been grown in culture for more than 60 passages. During this time, HUCB-NSCs retained their growth rate, the ability to differentiate into neuronal-, astrocyte-, and oligodendrocyte-like cells and displayed a stable karyotype. DNA microarray analysis of HUCB-NSCs revealed enhanced expression of selected genes encoding putative stem and progenitor cell markers when compared to other mononuclear cells. dBcAMP-induced HUCBNSCs were further differentiated into more advanced neuronal cells. This is the first report of the establishment and characterization of a nontransformed HUCB-NSC line that can be grown continuously in a monolayer culture and induced to terminal differentiation. These cells should further our understanding of the regulatory mechanisms involved in NSC self-renewal and differentiation.     

Massively parallel signature sequencing profiling of fetal human neural precursor cells

We have examined gene expression in multipotent neural precursor cells (NPCs) derived from human fetal (f) brain tissue and compared its expression profiles with embryonic stem (ESC) cells, embryoid body cell (EBC), and astrocyte precursors using the technique of massively parallel signature sequencing (MPSS). Gene expression profiles show that fNPCs express core neural stem cells markers and share expression profiles with astrocyte precursor cells (APCs) rather than ESC or EBC. Gene expression analysis shows that fNPCs differ from other adult stem and progenitor cells in their marker expression and activation of specific functional networks such as the transforming growth factorbeta (TGFbeta) and Notch signaling pathways. In addition, our results allow us to identify novel genes expressed in fNPCs and provide a detailed profile of fNPCs.     

Differentiation of Human Epidermal Neural Crest Stem Cells (hEPI-NCSC) into Virtually Homogenous Populations of Dopaminergic Neurons

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson’s disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFβ receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC.     

大鼠胚胎神经干细胞与永生化神经干细胞系C17.2定向分化为神经元潜能的差异

目的 对比大鼠早期胚胎神经干细胞( NSCs)与永生化神经干细胞系C17.2(C17.2-NSC)定向分化为神经元潜能的差异,确立适用于诱导NSCs定向分化为神经元高内涵药物筛选的NSCs筛选模型. 方法 C17.2-NSC、17d胚胎海马NSCs(E17-NSC)及11d胚胎大脑皮层NSCs(E11-NSC)均设定对照组和实验组,对照组与实验组的细胞在含有2％ (v/v) B27的DMEM/F12培养液中,分别经0μmol/L和1μmol/L维甲酸(RA)在37℃、5％CO2常规培养条件下诱导分化5d,通过免疫组织化学技术检测对照组与实验组中NSCs或前体细胞特异性标志蛋白巢蛋白(Nestin)及神经元特异性标志蛋白βⅢ微管蛋白(Tuj1)表达量的差异. 结果 与对照组相比,C17.2-NSC经1μμmol/L RA诱导后未定向分化为神经元,而E17-NSC及E11-NSC经1μmol/L RA诱导后可有效定向分化为神经元. 结论 相对于C17.2-NSC,大鼠早期胚胎NSCs定向分化为神经元的潜能更强,适用于诱导NSCs定向分化为神经元的高内涵药物筛选.     

Regulation of neural stem/progenitor cell maintenance by PI3K and mTOR

Control of stem cell state and differentiation of neural stem/progenitor cells is essential for proper development of the nervous system. EGF and FGF2 play important roles in the control of neural stem/progenitor cells, but the underlying mechanism still remains unclear. Here we show, using in vitro primary cultures of mouse neural stem/progenitor cells, that both PI3K and mTOR are activated by EGF/FGF2 but that inhibiting the activation of either PI3K or mTOR alone results in only reduced proliferation of neural stem/progenitor cells without affecting their stem cell state, namely, the capacity to self-renew. However, significantly, concurrent inhibition of PI3K and mTOR promoted exit from the stem cell state together with astrocytic differentiation of neural stem/progenitor cells. These findings suggest that PI3K and mTOR are involved in the EGF/FGF2-mediated maintenance of neural stem/progenitor cells and that they may act in parallel and independent pathways, complementing and backing up each other to maintain the stem cell state.     

Multilineage differentiation and characterization of the human fetal osteoblastic 1.19 cell line: a possible in vitro model of human mesenchymal progenitors

The in vitro study of human bone marrow mesenchymal stromal cells (BMMSCs) has largely depended on the use of primary cultures. Although these are excellent model systems, their scarcity, heterogeneity, and limited lifespan restrict their usefulness. This has led researchers to look for other sources of MSCs, and recently, such a population of progenitor/stem cells has been found in mesodermal tissues, including bone. We therefore hypothesized that a well-studied and commercially available clonal human osteoprogenitor cell line, the fetal osteoblastic 1.19 cell line (hFOB), may have multilineage differentiation potential. We found that undifferentiated hFOB cells possess similar cell surface markers as BMMSCs and also express the embryonic stem cell-related pluripotency gene, Oct-4, as well as the neural progenitor marker nestin. hFOB cells can also undergo multilineage differentiation into the mesodermal lineages of chondrogenic and adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Moreover, as with BMMSCs, under neural-inducing conditions, hFOB cells acquire a neural-like phenotype. This human cell line has been a widely used model of normal osteoblast differentiation. Our data suggest that hFOB cells may provide for researchers an easily available, homogeneous, and consistent in vitro model for study of human mesenchymal progenitor cells.     

An attempt to generate neurons from an astrocyte progenitor cell line FBD-104

In the present study, a clonal astrocyte progenitor cell line derived from p53-deficient fetal brains, named FBD-104, was characterized in monolayer and suspension culture. In monolayer culture with medium containing 10% serum, FBD-104 cells expressed some markers of astrocytes, such as glial fibrillary acidic protein (GFAP), S100beta, and glutamate aspartate transporter (GLAST). They never expressed any markers of neurons or oligodendrocytes. Thus the cell line appears to be restricted to the astroglial lineage. However, in suspension culture in serum-free medium supplemented with EGF and FGF2, FBD-104 cells proliferated and formed neurospheres expressing mRNAs for Mash1 and Math3, generating cells expressing neuron specific beta-III tubulin. Re-plating the spheres onto an adhesive substrate and withdrawal of the growth factors induced the expression of mRNAs for NeuroD and Olig2 and generated more beta-III tubulin-positive cells. The present study demonstrated that neurosphere culture is an efficient method to induce neurogenesis from the astrocyte progenitor cell line FBD-104. We also determined that pretreatment with FGF2 caused a significant increase in yield of neurospheres. Thus, the FBD-104 line is an interesting in vitro model to study effect of trophic factors and adhesive substrates on lineage determination of neural progenitor cells.     

Dopaminergic differentiation of the Nurr1-expressing immortalized mesencephalic cell line CSM14.1 in vitro

The use of neural stem cells as grafts is a potential treatment for Parkinson's disease, but the potential of stem cells to differentiate into dopaminergic neurones requires investigation. The present study examined the in vitro differentiation of the temperature-sensitive immortalized mesencephalic progenitor cell line CSM14.1 under defined conditions. Cells were derived from the mesencephalic region of a 14-day-old rat embryo, retrovirally immortalized with the Large T antigen and cultured at 33 degrees C in DMEM containing 10% fetal calf serum (FCS). For differentiation, the temperature was elevated at 39 degrees C and FCS was reduced (1%). Using histology, immunocytochemical detection of the stem cell marker Nestin and the neuronal marker MAP5 and, in addition, Western blotting to determine the presence of neurone-specific enolase and the neurone nuclei antigen we demonstrated a differentiation of these cells into neuronal cells accompanied by a decrease in Nestin production. In Western blots, we detected the orphan nuclear receptor Nurr1 in these cells. This was followed by a time-dependent up-regulation of the enzymes tyrosine hydroxylase and aldehyde dehydrogenase 2 characteristic of mature dopaminergic neurones. Our in vitro model of dopaminergic cell differentiation corroborates recent in vivo observations in the developing rodent brain.     

Glutamate enhances survival and proliferation of neural progenitors derived from the subventricular zone

Extracellular glutamate levels increase as a consequence of perinatal hypoxia/ischemia, causing the death of neurons and oligodendrocytes. Precursors in the subventricular zone (SVZ) also die following perinatal hypoxia/ischemia; therefore we hypothesized that glutamate would stimulate the death of neural precursors. Here we demonstrate using calcium imaging that SVZ derived neural stem/progenitor cells respond to both ionotropic and metabotropic excitatory amino acids. Therefore, we tested the effects of high levels of glutamate receptor agonists on the proliferation, survival, and differentiation of SVZ derived neural stem/progenitor cells in vitro. We show that high levels of glutamate, up to 1 mM, are not toxic to neural precursor cultures. In fact, stimulation of either the kainate receptor or group 2 metabotropic glutamate receptors (group 2 mGluR) reduces basal levels of apoptosis and increases neural precursor proliferation. Furthermore, group 2 mGluR activation expands the number of multipotent progenitor cells present in these cultures while maintaining equivalent mature cell production. We conclude that the glutamate released following perinatal hypoxia/ischemia may act to acutely promote the proliferation of multipotent precursors in the subventricular zone.     

Neuroprotective Effect of Mesenchymal and Neural Stem and Progenitor Cells on Sensorimotor Recovery after Brain Injury

We studied the effect of systemic administration of multipotent stem cells on impaired neurological status in rats with brain injury. It was found that transplantation of multipotent mesenchymal stromal cells of the bone marrow or human neural stem and progenitor cells to rats with local brain injury promoted recovery of the brain control over locomotor function and proprioceptive sensitivity of forelegs. The dynamics of neurological recovery was similar after transplantation of fetal neural stem and progenitor cells and multipotent mesenchymal stromal cells. Transplantation of cell cultures improved survival of experimental animals. It should be noted that administration of neural stem and progenitor cells prevented animal death not only in the acute traumatic period, but also in delayed periods.     

Neurofibromatosis-1 regulation of neural stem cell proliferation and multilineage differentiation operates through distinct RAS effector pathways

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by impaired function of the neurofibromin RAS regulator. Using a combination of Nf1 genetically engineered mice and pharmacological/genetic inhibition approaches, we report that neurofibromin differentially controls neural stem cell (NSC) proliferation and multilineage differentiation through the selective use of the PI3K/AKT and RAF/MEK pathways. While PI3K/AKT governs neurofibromin-regulated NSC proliferation, multilineage differentiation is MEK-dependent. Moreover, whereas MEK-regulated multilineage differentiation requires Smad3-induced Jagged-1 expression and Notch activation, MEK/Smad3-regulated Hes1 induction is only responsible for astrocyte and neuronal differentiation. Collectively, these findings establish distinct roles for the RAS effector pathways in regulating brain NSC function.