One Mouse,Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.     

Effects of sulforaphane on neural stem cell proliferation and differentiation

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

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis,Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.     

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

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.     

低糖低氧对神经干细胞增殖和代谢的影响

目的:本研究旨在探讨低糖低氧对大鼠神经干细胞增殖和代谢的影响。方法:实验采用不同葡萄糖浓度的培养基以及不同的氧浓度进行处理:高糖(4.5g/L)、低糖(1.4g/L);常氧(20%O2)、低氧(3%O2);神经干细胞(NSCs)来自孕13.5d的大鼠中脑,在不同糖浓度下培养至第三代进行低氧处理,分为低糖常氧(L+N)、低糖低氧(L+H)、高糖常氧(H+N)、高糖低氧(H+H)组。神经干细胞在上述四种条件下分别培养1、3、5d后,利用CCK-8检测神经干细胞的增殖情况;生化分析仪测定细胞培养上清液中葡萄糖、乳酸、丙酮酸浓度;RT-PCR方法检测葡萄糖转运蛋白4(GluT4)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)的表达变化。结果:在低糖低氧条件下培养3d时NSCs的数量增加最为明显;低糖低氧条件下,葡萄糖浓度降低最为显著;而丙酮酸浓度在低糖处理组均高于高糖处理组;同样地,在低糖低氧处理组培养上清中乳酸含量增加的幅度最大;此外,在低糖或低氧时Glut4和PK的表达也明显高于对照组。结论:低氧能促进NSCs的增殖,而以低氧和低糖共同作用时更为明显;在低氧低糖条件下,神经干细胞的代谢发生变化,葡萄糖的利用明显增加,主要通过糖酵解途径代谢产能。     

Strengths and limitations of the neurosphere culture system

After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a nearperfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.     

Role of Cyclic Nucleotide-Gated Channels in the Modulation of Mouse Hippocampal Neurogenesis

Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.     

Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.     

Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon

Multipotent, self-renewing neural stem cells reside in the embryonic mouse telencephalic germinal zone. Using an in vitro neurosphere assay for neural stem cell proliferation, we demonstrate that FGF-responsive neural stem cells are present as early as E8.5 in the anterior neural plate, but EGF-responsive neural stem cells emerge later in development in a temporally and spatially specific manner. By separately blocking EGF and FGF2 signaling, we also show that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation and at relatively high cell densities separate cell nonautonomous effects can substantially enhance the mitogen-induced proliferation. At lower cell densities, neural stem cell proliferation is additive in the presence of EGF and FGF2 combined, revealing two different stem cell populations. However, both FGF-responsive and EGF-responsive neural stem cells retain their self-renewal and multilineage potential, regardless of growth factor conditions. These results support a model in which separate, lineage-related EGF- and FGF-responsive neural stem cells are present in the embryonic telencephalic germinal zone.     

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM ^SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM ^SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM ^SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM ^SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.     

Determination of somatic and cancer stem cell self-renewing symmetric division rate using sphere assays

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cell's resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence.     

Effects of the monomeric, oligomeric, and fibrillar Abeta42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone

The incidence of amyloid plaques, composed mainly of beta-amyloid peptides (Abeta), does not correlate well with the severity of neurodegeneration in patients with Alzheimer's disease (AD). The effects of Abeta(42) on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Abeta(42) peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 micromol/L) of oligomeric Abeta(42) increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Abeta(42) on NSCs in the brains of patients with AD.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.     

远志皂苷对丙泊酚麻醉所致大鼠认知功能障碍的保护作用及机制

本研究旨在探讨远志皂苷对丙泊酚麻醉所致大鼠认知功能障碍的保护作用及机制。将SD大鼠分别应用远志皂苷(200 mL·kg^-1·d^-1)和/或丙泊酚(60 mL·kg^-1·d^-1)处理3周,通过Morris水迷宫实验来评价认知功能情况,通过苏木精伊红(hematoxylineosin, HE)染色评价组织病理改变。采用原位末端标记技术(TdTmediated dUTP nick and labeling, Tunel)检测大鼠海马神经细胞的凋亡情况。采用酶联免疫吸附法(enzymelinked immunosorbent assay, ELISA)检测氧化损伤指标。采用Western blotting检测Bcl-2和Caspase-3的表达。Morris水迷宫实验显示,远志皂苷显著降低了丙泊酚麻醉大鼠的逃避潜伏期,并提高了穿越平台次数(p<0.05)。苏木精伊红(HE)染色显示,远志皂苷可显著降低丙泊酚引起的大鼠海马组织病变程度。原位末端标记技术(Tunel)实验显示,远志皂苷抑制了丙泊酚引起的大鼠海马神经细胞凋亡(p<0.05)。Western blotting检测显示,远志皂苷抑制了丙泊酚对大鼠脑组织中Bcl-2蛋白的下调及Caspase-3的上调(p<0.05)。酶联免疫吸附检测显示,远志皂苷提高了大鼠脑组织中SOD和GSH的水平,并降低了MDA水平(p<0.05)。远志皂苷可显著改善丙泊酚麻醉大鼠的认知功能并降低海马组织病变程度,其机制与抑制海马神经细胞凋亡和减弱氧化损伤有关。     

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.     

The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival

5-Bromo-2'-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48?h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.