Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process

We examined the role of Notch signaling on the generation of neurons and glia from neural stem cells by using neurospheres that are clonally derived from neural stem cells. Neurospheres prepared from Dll1(lacZ/lacZ) mutant embryos segregate more neurons at the expense of both oligodendrocytes and astrocytes. This mutant phenotype could be rescued when Dll1(lacZ/lacZ) spheres were grown and/or differentiated in the presence of conditioned medium from wild-type neurospheres. Temporal modulation of Notch by soluble forms of ligands indicates that Notch signaling acts in two steps. Initially, it inhibits the neuronal fate while promoting the glial cell fate. In a second step, Notch promotes the differentiation of astrocytes, while inhibiting the differentiation of both neurons and oligodendrocytes.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

A Modified In vitro Method to Obtain Pure Astrocyte Cultures Induced from Mouse Hippocampal Neural Stem Cells Using Clonal Expansion

The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus, which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells, which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded 94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase. Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture of highly purified astrocytes from mouse hippocampus.     

Symposium 10: Differentiation Plasticity of Stem Cells. Direct differentiation of radial glial cells from embryonic stem cells

The major role of radial glial cells in neuronal development is to provide support and guidance for neuronal migration. In vitro, neurons, astrocytes and oligodendrocytes have also been generated from neural stem cells and embryonic stem cells, but the generation of radial glial cells in vitro has not yet been reported. Since radial glial cells can lead to neurons and astrocytes during brain development, neurogenesis and gliogenesis of stem cells in vitro may at least in part also utilize the same mechanisms. To test this hypothesis, we utilized five different clones of embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glial cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glial cells are similar in morphology to radial glial cells in vivo. They also express several cytoskeletal markers that are characteristics of radial glial cells in vivo. The processes of these in vitro‐generated radial glial cells are organized into scaffolds that appear to support the migration of newly generated neurons in culture. Like radial glial cells in vivo, they appear to differentiate subsequently into astrocytes. Differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system may facilitate the investigation of regulation of radial glial cell differentiation and its biological function. Acknowledgements: Supported by USPHS Grant NS11853 and a grant from the Children's Medical Research Foundation.     

神经干细胞向神经元分化调控的研究进展

神经干细胞是一类具有分裂潜能和自更新能力的母细胞,它可以通过对称分裂和不对称分裂方式产生神经组织的各类细胞,包括神经元、星形胶质细胞和少突胶质细胞。中枢神经系统受到损伤后,神经元和胶质细胞的损伤导致了临床症状,内源性神经干细胞的修复作用不大,原因是干细胞的数量有限,微环境的不允许。移植的神经干细胞进入体内后,由于受到多种因素的影响,常保持未分化状态或大部分分化为胶质细胞。神经干细胞向神经元分化的调控机制及其影响因素直接决定神经干细胞源性神经元的比例和神经元之间功能性突触的数量。现就其研究进展做一综述。     

Neural stem cells in the brain

Stem cells in the central nervous system were usually considered as relevant for evaluation only in embryonic time. Recent advances in molecular cloning and immunological identification of the different cell types prove the presence of neurogenesis of the new neurons in adult mammals brains. New neurons are born in two areas of the mammal and human brain--sybventricular zone and subgranular zone of dentate gyrus. New born granular neurons of dentate gyrus have a great importance for memory and learning. New neurons originate from precursors which in culture and in situ could also transform into astrocytes and oligodendrocytes, thus fulfill criteria of neural stem cells. In culture, mitotic activity of these stem sells depends on fibroblast growth factor 2 and epidermal growth factor. Depletion of cultural medium of these factors and addition of serum, other growth factors (Platelet-derived growth factor and ciliary neurotrophic factor) leads to generation of neurons and astrocytes. Isolation and clonal analysis of stem cells is based on immunological markers such as nestin, beta-tubulin III, some types of membrane glicoproteids. Identification and visualization of stem cells in brain revealed two populations of cells which have properties of stem cells. In embryonic time, radial glia cells could give origin to neurons, in mature brain cells expressing glial fibrillar acidic protein typical marker of astrocytes fulfill criteria for stem cells. Neural stem cells could transform not only into mature neurons and glial cells but also into blood cells, thus revealing broad spectrum of progenitors from different embryonic tissues. Further progress in this field of neurobiology could give prosperity in the cell therapy of many brain diseases.     

Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.     

Direct reprogramming of human astrocytes into neural stem cells and neurons

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for neurological repair. Here we show dedifferentiation of human cortical astrocytes into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate. Ectopic expression of the reprogramming factors OCT4, SOX2, or NANOG into astrocytes in specific cytokine/culture conditions activated the neural stem gene program and induced generation of cells expressing neural stem/precursor markers. Pure CD44+ mature astrocytes also exhibited this lineage commitment change and did not require passing through a pluripotent state. These astrocyte-derived neural stem cells gave rise to neurons, astrocytes, and oligodendrocytes and showed in vivo engraftment properties. ASCL1 expression further promoted neuronal phenotype acquisition in vitro and in vivo. Methylation analysis showed that epigenetic modifications underlie this process. The restoration of multipotency from human astrocytes has potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders.     

Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity.     

Isolation and characterization of neural stem cells from the neonatal rat cochlear nucleus

Neural stem cells have been identified in multiple parts of the postnatal mammalian brain, as well as in the inner ear. No investigation of potential neural stem cells in the cochlear nucleus has yet been performed. The aim of this study was to investigate potential neural stem cells from the cochlear nucleus by neurosphere assay and in histological sections to prove their capacity for self-renewal and for differentiation into progenitor cells and cells of the neuronal lineage. For this purpose, cells of the cochlear nucleus of postnatal day 6 rats were isolated and cultured for generation of primary neurospheres. Spheres were dissociated and cells analyzed for capacity for mitosis and differentiation. Cell division was detected by cell-counting assay and BrdU incorporation. Differentiated neural progenitor cells showed distinct labeling for Nestin and for Atoh1. Positive staining of ß-III Tubulin, glial fibrillary acid protein (GFAP) and myelin basic protein (MBP) showed differentiation into neurons, astrocytes and oligodendrocytes. Furthermore, Nestin- and BrdU-labeled cells could also be detected in histological sections. In conclusion, the isolated cells from the cochlear nucleus presented all the features of neural stem cells: cell division, presence of progenitor cells and differentiation into different cells of the neuronal lineage. The existence of neural stem cells may add to the understanding of developmental features in the cochlear nucleus.     

Isolation and characterization of neural stem cells from human fetal striatum

This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience.     

Phosphatidylglucoside: a novel marker for adult neural stem cells

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.     

Neural stem cells in the adult human brain

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.     

Cellular Environment Directs Differentiation of Human Umbilical Cord Blood�CDerived Neural Stem Cells In Vitro

Cord blood–derived neural stem cells (NSCs) are proposed as an alternative cell source to repair brain damage upon transplantation. However, there is a lack of data showing how these cells are driven to generate desired phenotypes by recipient nervous tissue. Previous research indicates that local environment provides signals driving the fate of stem cells. To investigate the impact of these local cues interaction, the authors used a model of cord blood–derived NSCs co-cultured with different rat brain–specific primary cultures, creating the neural-like microenvironment conditions in vitro. Neuronal and astro-, oligo-, and microglia cell cultures were obtained by the previously described methods. The CMFDA-labeled neural stem cells originated from, non-transformed human umbilical cord blood cell line (HUCB-NSCs) established in a laboratory. The authors show that the close vicinity of astrocytes and oligodendrocytes promotes neuronal differentiation of HUCB-NSCs, whereas postmitotic neurons induce oligodendrogliogenesis of these cells. In turn, microglia or endothelial cells do not favor any phenotypes of their neural commitment. Studies have confirmed that HUCB-NSCs can read cues from the neurogenic microenvironment, attaining features of neurons, astrocytes, or oligodendrocytes. The specific responses of neurally committed cord blood–derived cells, reported in this work, are very much similar to those described previously for NSCs derived from other “more typical” sources. This further proves their genuine neural nature. Apart from having a better insight into the neurogenesis in the adult brain, these findings might be important when predicting cord blood cell derivative behavior after their transplantation for neurological disorders.     

Pro-regenerative properties of cytokine-activated astrocytes

The prevailing view of the astrocytic response to injury is that reactive astrocytes impede the regenerative process by forming scar tissue. As the levels of many cytokines dramatically increase following CNS insult and as this increase in cytokine expression precedes the production of the glial scar, a long-standing view has been that cytokines diminish neuronal survival and regeneration by stimulating the formation of astrogliotic scar tissue. However, there is a wealth of data indicating that cytokines "activate" astrocytes, and that cytokine-stimulated astrocytes can promote the recovery of CNS function. Supporting evidence demonstrates that cytokine-activated astrocytes produce energy substrates and trophic factors for neurons and oligodendrocytes, act as free radical and excess glutamate scavengers, actively restore the blood-brain barrier, promote neovascularization, restore CNS ionic homeostasis, promote remyelination and also stimulate neurogenesis from neural stem cells. Accordingly, a re-assessment of cytokine-activated astrocytes is necessary. Here, we review studies that promote the thesis that cytokines elicit potent neuroprotective and regenerative responses from astrocytes.     

神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

肿瘤坏死因子-α对大鼠中脑神经干细胞分化和寡突胶质细胞增殖的影响

为观察肿瘤坏死因子对神经干细胞(NSCs)分化的影响,本研究应用体外扩增的新生大鼠中脑NSCs,使用免疫组织化学技术,观察了肿瘤坏死因子—α(TNF—α)对NSCs分化及其后代细胞的影响。结果显示：(1)TNF—α可提高中脑NSCs后代中神经元和寡突胶质细胞所占的比例;(2)TNF—α可明显诱导由NSCs分化的寡突胶质细胞增殖,但对星形胶质细胞的增殖作用不明显。上述观察结果提示TNF—α对NSCs的应用具有重要影响。     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.