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PubMed2006

Differentiation profile of brain tumor stem cells: a comparative study with neural stem cells.

Understanding of the differentiation profile of brain tumor stem cells (BTSCs), the key ones among tumor cell population, through comparison with neural stem cells (NSCs) would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and beta-tubulinIII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.

AC133 Antigen↗
PubMed2002

[Studies of effects of lipid on rat fetal neural stem cells].

Rat fetal neural stem cells (rFNSCs) was separated from embryo about 14.5-16.5 days, and cultured in DMEM/F12 media with additives and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Effects of lipid on growth and proliferation of rFNSCs was examined by counting the number of neurospheres and incorporation of 3H. The data show that chemical defined lipid improved rFNSCs' growth and cell division. Lipid will be another neural stem cell's culture media's additive.

English Abstract↗
PubMed2003

Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro.

In some rodent models, there is evidence that hematopoietic stem cells (HSC) can differentiate into neural cells. However, it is not known whether humans share this potential, and, if so, what conditions are sufficient for this transdifferentiation to occur. We addressed this question by assessing the ability of fetal human liver CD34(+)/CD133(+)/CD3(-) hematopoietic stem cells to generate neural cells and astrocytes in culture. We cultured fetal liver-derived hematopoietic stem cells in human astrocyte culture-conditioned medium or using a method wherein growing human astrocytes were separated from cultured, nonadherent hematopoietic stem cells by a semipermeable membrane in a double-chamber co-culture system. Hematopoietic stem cell cultures were probed for neural progenitor cell marker expression (nestin and bone morphogenic protein-2 [BMP-2]) during growth in both culture conditions. RT-PCR, western blotting, and immunocytochemistry assays showed that cells cultured in either condition expressed nestin mRNA and protein and BMP-2 mRNA. HSC similarly cultured in nonconditioned medium or in the absence of astrocytes did not express either marker. Cells expressing these neural markers were transferred and cultured on poly-D-lysine-coated dishes with nonconditioned growth medium for further study. Immunocytochemistry demonstrated that these cells differentiated into astrocytes after 8 days in culture as indicated by their morphology and expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and S100, as well as by their rate of proliferation, which was identical to that of freshly isolated fetal brain astrocytes. These findings demonstrate that neural precursor gene expression can be induced when human hematopoietic stem cells are exposed to a suitable microenvironment. Furthermore, the neural stem cells generated in this environment can then differentiate into astrocytes. Therefore, human hematopoietic stem cells may be an alternative resource for generation of neural stem cells for therapy of central nervous system defects resulting from disease or trauma.

AC133 Antigen↗
PubMed2002

Developmental changes in Notch1 and numb expression mediated by local cell-cell interactions underlie progressively increasing delta sensitivity in neural crest stem cells.

Neural stem cells become progressively less neurogenic and more gliogenic with development. Here, we show that between E10.5 and E14.5, neural crest stem cells (NCSCs) become increasingly sensitive to the Notch ligand Delta-Fc, a progliogenic and anti-neurogenic signal. This transition is correlated with a 20- to 30-fold increase in the relative ratio of expression of Notch and Numb (a putative inhibitor of Notch signaling). Misexpression experiments suggest that these changes contribute causally to increased Delta sensitivity. Moreover, such changes can occur in NCSCs cultured at clonal density in the absence of other cell types. However, they require local cell-cell interactions within developing clones. Delta-Fc mimics the effect of such cell-cell interactions to increase Notch and decrease Numb expression in isolated NCSCs. Thus, Delta-mediated feedback interactions between NCSCs, coupled with positive feedback control of Notch sensitivity within individual cells, may underlie developmental changes in the ligand-sensitivity of these cells.

Animals↗
PubMed2006

[A comparison study for differentiation between human fetal retinal progenitor cells and brain neural stem cells in vitro].

OBJECTIVE: Investigating the potential of differentiation of human fetal retinal progenitor cells (hRPCs) and brain neural stem cells (hBNSCs) in vitro. METHODS: hRPCs and hBNSCs were isolated from human fetuses (8-12 weeks of gestation) and cultured in serum-free DMEM/F12 culture medium with N2 supplement, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) or culture medium with 10% fetal bovine serum (FBS) but without EGF and bFGF. Immunocytochemical and immunofluorescence studies were conducted for identification of neural stem cells, retinal progenitors or the subtypes of neurons, astrocytes, retinal ganglion cells and rod photoreceptors with the specific antibodies for Nestin, Pax6, Map2, GFAP, Thy-1 and Rhodopsin, respectively. RESULTS: Both hRPCs and hBNSCs could proliferate and differentiate in DMEM/F12 + N2 with or without 10% FBS and expressed specific markers of immature neuroepithelial cells, retinal progenitors, mature neurons, astrocytes, retinal ganglion cells and rod photoreceptors. hBNSCs easily attached, spread out longer neurites and to form a network when cultured with serum contained medium. hRPCs were more difficult to attach and had only short dendrites. CONCLUSIONS: Both hRPCs and hBNSCs can differentiate into retinal specific cell types in vitro. The adherent, migration and differential capacity of hRPCs and hBNSCs are different when these cells are induced by the serum-contained culture medium.

Brain↗
PubMed2006

Potential role of NF-kappaB in adult neural stem cells: the underrated steersman?

Neural stem cells are precursors of neurons and glial cells. During brain development, these cells proliferate, migrate and differentiate into specific lineages. Recently neural stem cells within the adult central nervous system were identified. Informations are now emerging about regulation of stem cell proliferation, migration and differentiation by numerous soluble factors such as chemokines and cytokines. However, the signal transduction mechanisms downstream of these factors are less clear. Here, we review potential evidences for a novel central role of the transcription factor nuclear factor kappa B (NF-kappaB) in these crucial signal transduction processes. NF-kappaB is an inducible transcription factor detected in neurons, glia and neural stem cells. NF-kappaB was discovered by David Baltimore's laboratory as a transcription factor in lymphocytes. NF-kappaB is involved in many biological processes such as inflammation and innate immunity, development, apoptosis and anti-apoptosis. It has been recently shown that members of the NF-kappaB family are widely expressed by neurons, glia and neural stem cells. In the nervous system, NF-kappaB plays a crucial role in neuronal plasticity, learning, memory consolidation, neuroprotection and neurodegeneration. Recent data suggest an important role of NF-kappaB on proliferation, migration and differentiation of neural stem cells. NF-kappaB is composed of three subunits: two DNA-binding and one inhibitory subunit. Activation of NF-kappaB takes place in the cytoplasm and results in degradation of the inhibitory subunit, thus enabling the nuclear import of the DNA-binding subunits. Within the nucleus, several target genes could be activated. In this review, we suggest a model explaining the multiple action of NF-kappaB on neural stem cells. Furthermore, we discuss the potential role of NF-kappaB within the so-called brain cancer stem cells.

Animals↗
PubMed2003

Generation of functional radial glial cells by embryonic and adult forebrain neural stem cells.

Radial glial cells (RGCs), a transient cell population present only in the developing CNS, function both as precursor cells and as scaffolds to support neuron migration. Their cellular origin, however, is not understood. In the present study, we tested the hypothesis that functional RGCs can be generated by multipotent neural stem cells. Embryonic forebrain neural stem cells were studied in vitro to identify putative signals that promote the generation and differentiation of functional RGCs, determined by their ability to support neuronal migration. Epidermal growth factor receptor signaling was sufficient to regulate both the generation and differentiation of morphologically, antigenically, and functionally defined RGCs. In contrast, fibroblast growth factor-2 promoted the generation of RGCs but was unable to support their differentiation. Although RGCs are not normally present in the adult brain, epidermal growth factor stimulated adult forebrain neural stem cells to generate RGCs in vitro and functional RGCs within the adult forebrain subependyma in vivo. Surprisingly, epidermal growth factor receptor signaling also promoted adult forebrain ependymal cells to dedifferentiate and adopt a radial morphology in vivo. These results suggest that neural stem cells can give rise to RGCs and that RGC-guided neuronal migration can be recapitulated in the adult CNS.

Animals↗
PubMed2004

Disguising adult neural stem cells.

A description of adult neural stem cells has remained somewhat elusive. With no unique and definitive markers to label stem cells in general, neural stem cells are difficult to identify definitively and one is forced to examine cell behavior - leading to the retrospective identification of a stem cell. The most prevalent view in the literature describes the adult forebrain neural stem cell as a relatively quiescent subependymal cell that expresses glial fibrillary acidic protein. In this review we summarize some of the recent studies that have supported or challenged this conception of the nature of the adult forebrain neural stem cell. We highlight and compare the experimental paradigms that have led to our current state of knowledge and conclude that there is no reason at present to abandon the prevailing view of adult neural stem cell identity.

Animals↗
PubMed2003

Neural stem cells in the adult nervous system.

The concept of the immutability of the nervous tissue has recently been replaced with the new idea that a continuous neurogenic turnover does occur in some limited areas of the central nervous system (CNS). At least two neurogenic regions of the adult mammalian CNS are involved in this process: the subventricular zone of the forebrain and the dentate gyrus of the hippocampus, which are considered to be a reservoir of new neural cells. Neural stem cells (NSCs) are multipotential progenitors that have self-renewal capability. While in vivo endogenous NSCs seem able to produce almost exclusively neurons, a single NSC in vitro is competent to generate neurons, astrocytes, and oligodendrocytes. NSCs lack a specific morphology and unambiguous surface markers that could allow their identification. For this reason, one of the major difficulties in identifying stem cells is that they are defined in terms of their functional capabilities, the determination of which might alter the cells' nature. The purpose of this review is to describe the characteristics of the NSCs of the adult mammalian CNS, their potentiality in terms of proliferation and differentiation capabilities, as well as their stability in long-term culture, all attributes that make them a good tool for tissue replacement therapies.

Adult↗
PubMed2002

Co-transfection with cDNA encoding the Bcl family of anti-apoptotic proteins improves the efficiency of transfection in primary fetal neural stem cells.

Inefficiency in primary neural stem cell transfection is a major obstacle in gene expression research aimed at determining the mechanisms underlying brain development. Following our initial finding that liposome-mediated transfection procedures are fairly toxic to neural stem cells, we further examined whether transfection efficiency could be improved by preventing cells from undergoing apoptosis. Transfection efficiencies were markedly enhanced by co-transfection of cells with prototypic anti-apoptotic genes, such as bcl-2 and bcl-xL, and supplementing the culture medium with B27 Supplement. This combination of anti-apoptotic gene co-transfection and B27 Supplement resulted in approximately 5% transfection efficiency of primary neural stem cells, compared to less than 0.2% in control transfections. Therefore, this procedure and other similar approaches employed to enhance the efficiency of transfecting neural stem cells may facilitate the understanding of mechanisms underlying self-renewal of neural stem cells and their differentiation into various cell lineages.

Animals↗
PubMed2003

Interactions between human adipose stromal cells and mouse neural stem cells in vitro.

Transplantation of adult mesenchymal stem cells (MSCs) into adult rat brain has been known to reduce functional deficits associated with stroke and traumatic brain injury. However, in injured brains, there is no evidence that transplanted MSCs replace lost host brain tissue. In this study, we determined in vitro interaction between human adipose tissue stromal cells (hATSCs), a kind of MSC, and neural stem cells (NSCs). hATSCs were isolated and proliferated from human adipose tissues, and NSCs from the subventricular zone of postnatal mice. When NSCs were cultured on mitomycin-treated hATSC monolayers, their proliferation was decreased, but neuronal differentiation was significantly induced. The percentage of neurons significantly increased in 7 days in cultures of NSCs on hATSCs feeder as compared to NSCs cultured on laminin-coated dishes. When the duration of the cultures was extended to 14 days, hATSCs supported the survival of neurons derived from NSCs. To determine the role of soluble factors from hATSCs, NSCs were cultured with hATSCs conditioned medium or co-cultured with permeable filter on which hATSCs were grown. Although proliferation of NSCs significantly decreased and glial differentiation increased under these experimental conditions, their neuronal differentiation was not affected, indicating that direct physical contact between hATSCs and NSCs is required for induction of neuronal differentiation. These data indicate that hATSCs may provide supportive roles on endogenous neural stem cells, when they are transplanted into damaged brain.

Adipose Tissue↗
PubMed2006

ABC transporters, neural stem cells and neurogenesis--a different perspective.

Stem cells intrigue. They have the ability to divide exponentially, recreate the stem cell compartment, as well as create differentiated cells to generate tissues. Therefore, they should be natural candidates to provide a renewable source of cells for transplantation applied in regenerative medicine. Stem cells have the capacity to generate specific tissues or even whole organs like the blood, heart, or bones. A subgroup of stem cells, the neural stem cells (NSCs), is characterized as a self-renewing population that generates neurons and glia of the developing brain. They can be isolated, genetically manipulated and differentiated in vitro and reintroduced into a developing, adult or a pathologically altered central nervous system. NSCs have been considered for use in cell replacement therapies in various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Characterization of genes with tightly controlled expression patterns during differentiation represents an approach to understanding the regulation of stem cell commitment. The regulation of stem cell biology by the ATP-binding cassette (ABC) transporters has emerged as an important new field of investigation. As a major focus of stem cell research is in the manipulation of cells to enable differentiation into a targeted cell population; in this review, we discuss recent literatures on ABC transporters and stem cells, and propose an integrated view on the role of the ABC transporters, especially ABCA2, ABCA3, ABCB1 and ABCG2, in NSCs' proliferation, differentiation and regulation, along with comparisons to that in hematopoietic and other stem cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2004

Neural stem cell differentiation in a cell-collagen-bioreactor culture system.

Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia. Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels. The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits. However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived. In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks. Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors. Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week. The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production. After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures. In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites. These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy.

Animals↗
PubMed1999

Extended serial passaging of mammalian neural stem cells in suspension bioreactors.

Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.

Animals↗
PubMed2001

A new 'spin' on neural stem cells?

The existence of neural stem cells in the adult brain was essentially denied until the last decade. Within the past ten years, considerable progress has been made in examining the fundamental properties of neural stem cells. Most recently there has been much interest in the identification and precise location of the adult neural stem cells in vivo. Studies examining the localization of neural stem cells are controversial and suggest two distinct locations within the adult brain: the ependymal layer lining the ventricles, and the subependymal layer immediately adjacent to the ependyma.

Adult↗
PubMedDate not supplied

Blocking BE301622 gene expression by RNAi initiates differentiation of neural stem cells in rat.

Neural stem cells (NSCs) are capable of differentiating into neurons, astrocytes and oligodendrocytes. However, the molecular mechanisms regulating NSCs differentiation are not well understood. Our previous research by microarray analysis certified that a lot of genes are differentially expressed in the course of NSC differentiation. In this study we report the function of one of these genes, BE301622, by RNAi techniques. To silence the BE301622 gene, a long, double-stranded RNA (dsRNA) was synthesized by using a kit (Ambion T7 MegaScript) and transformed into NSCs. Expression of mRNA was tested through RT-PCR. The result showed the expression of BE301622 gene was specificially suppressed. This finding effectively validated that BE301622 is involved in the differentiation of NSCs.

Animals↗