Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Effect of Optogenetic Stimulus on the Proliferation and Cell Cycle Progression of Neural Stem Cells

Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G₁ phase and reduced the percentage in S phase. It is likely that the influence of blue light on cell proliferation and the cell cycle was mediated by membrane depolarization, which induced accumulation of p21 and p27 proteins. Our data provide additional specific evidence that membrane depolarization may inhibit neural stem cell proliferation.     

神经干细胞克隆球中干细胞的比例变化

为了定量研究神经干细胞体外产生的克隆结构“neurospheres”中干细胞的比例变化,利用无血清培养、细胞克隆培养技术及免疫细胞化学染色方法,观察不同代数神经干细胞克隆球中nestin阳性细胞的比例。发现随着传代次数增加,克隆球中nestin阳性细胞的比例也在显著减少(P<0.001)。提示在体外培养体系中,形成的克隆球具有异质性,并且在不同代数间神经干细胞的比例也显著不同。     

In vitro and in vivo characterization of neural stem cells

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.     

Culture of mouse neural stem cell precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types. This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.     

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.     

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.     

大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

大鼠脑神经干细胞系（RNSC—FMU 1）的建立和鉴定

A neural stem cell line (RNSC-FMU 1) from rat brain have been established successfully by isolating and culturing neural stem cells from newborn SD rat brain in vitro with free-serum medium and passaging with mechanical division. The cell line cultured can continuously generate in vitro for long-term and it is 21 months (>100 passages) so far. These cells keep the feature of neural stem cell and normal karyotype. These neural stem cells can be induced to differentiate into neurons, astrocytes and oligodendrocytes. The cells have an extensive self-renewal capacity; its doubling time of proliferation is about 20 h. The cells are also cryopreservable. Tumor formation is not observed in nude mice that explanted with the cells. This cell line is a good tool for research of neural stem cell.     

Glioma stem cells: a midterm exam

Several years ago, the discovery of a highly tumorigenic subpopulation of stem-like cells embedded within fresh surgical isolates of malignant gliomas lent support to a new paradigm in cancer biology--the cancer stem cell hypothesis. At the same time, these "glioma stem cells" seemed to resolve a long-standing conundrum on the cell of origin for primary cancers of the brain. However, central tenets of the cancer stem cell hypothesis have recently been challenged, and the cellular origins of stem-like cells within malignant glioma are still contended. Here, we summarize the issues that are still in play with respect to the cancer stem cell hypothesis, and we revisit the developmental origins of malignant glioma. Do glioma stem cells arise from developmentally stalled neural progenitors or from dedifferentiated astrocytes? Five separate predictions of a neural progenitor cell of origin are put to the test.     

Hypoxia and neural stem cells: from invertebrates to brain cancer stem cells

Oxygen is a fundamental element for all living organisms, and modifications in its concentration influence several physiological and pathological events such as embryogenesis, development and also aging. Regulation of oxygen levels is an important factor in neural stem cell biology (e.g. differentiation, growth and the capacity to generate more differentiated cells). Studies on neural stem cells in culture have deepened our knowledge of their survival, proliferation and differentiation pathways. However, traditional cell culture for neural stem cells is performed employing environmental oxygen levels of 20%, while the effective oxygen concentration in the developing and adult brain is significantly lower; this results in an important alteration of the in vivo conditions. Several data indicate that a so called "physiologic hypoxic condition" could strongly influence the growth of neural stem cells and their differentiation mechanisms both in vivo and in vitro. The present overview deals with the different mechanisms utilized by invertebrate and vertebrate organisms to respond to hypoxic conditions. It highlights how the adaptations and responses to different oxygen concentrations have changed along the developmental route and underlines the importance of oxygen concentration in neural physiology and differentiation, with a final hint to the involvement of hypoxia in brain cancer stem cells.     

CD133 (Prominin) Negative Human Neural Stem Cells Are Clonogenic and Tripotent

Background

CD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined.

Methodology/Principal Findings

In this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion.

Conclusions

The finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.     

Location, location, location: the cancer stem cell niche

The existence of a stem cell niche, or physiological microenvironment, consisting of specialized cells that directly and indirectly participate in stem cell regulation has been verified for mammalian adult stem cells in the intestinal, neural, epidermal, and hematopoietic systems. In light of these findings, it has been proposed that a "cancer stem cell niche" also exists and that interactions with this tumor niche may specify a self-renewing population of tumor cells. We discuss emerging data that support the idea of a veritable cancer stem cell niche and propose several models for the relationship between cancer cells and their niches.     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

TGF-beta in neural stem cells and in tumors of the central nervous system

Mechanisms that regulate neural stem cell activity in the adult brain are tightly coordinated. They provide new neurons and glia in regions associated with high cellular and functional plasticity, after injury, or during neurodegeneration. Because of the proliferative and plastic potential of neural stem cells, they are currently thought to escape their physiological control mechanisms and transform to cancer stem cells. Signals provided by proteins of the transforming growth factor (TGF)-beta family might represent a system by which neural stem cells are controlled under physiological conditions but released from this control after transformation to cancer stem cells. TGF-beta is a multifunctional cytokine involved in various physiological and patho-physiological processes of the brain. It is induced in the adult brain after injury or hypoxia and during neurodegeneration when it modulates and dampens inflammatory responses. After injury, although TGF-beta is neuroprotective, it may limit the self-repair of the brain by inhibiting neural stem cell proliferation. Similar to its effect on neural stem cells, TGF-beta reveals anti-proliferative control on most cell types; however, paradoxically, many brain tumors escape from TGF-beta control. Moreover, brain tumors develop mechanisms that change the anti-proliferative influence of TGF-beta into oncogenic cues, mainly by orchestrating a multitude of TGF-beta-mediated effects upon matrix, migration and invasion, angiogenesis, and, most importantly, immune escape mechanisms. Thus, TGF-beta is involved in tumor progression. This review focuses on TGF-beta and its role in the regulation and control of neural and of brain-cancer stem cells. This work was supported by the German Federal Ministry of Education and Research (BMBF no. 01GA0510 and no. 0312134) and by the Bavarian State Ministry of Sciences, Research and the Arts, "Forneurocell grant".     

Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus

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Highlights? The neural stem cell pool declines over time during aging ? Neural stem cell activation prompts rapid asymmetric divisions ? After undergoing division, neural stem cells differentiate into astrocytes     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.