Neural progenitors derived from monkey embryonic stem cells in a simple monoculture system

A simple monoculture system. combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditio

Directed Differentiation of Neural Progenitors into Neurons Is Accompanied by Altered Expression of P2X Purinergic Receptors

Neural differentiation has been extensively studied in vitro in a model termed neurospheres, which consists of aggregates of neural progenitor cells. Previous studies suggest that they have a great potential for the treatment of neurological disorders. One of the major challenges for scientists is to control cell fate and develop ideal culture conditions for neurosphere expansion in vitro, without altering their features. Similar to human neural progenitors, rat neurospheres cultured in the absence of epidermal and fibroblast growth factors for a short period increased the levels of beta-3 tubulin and decreased the expression of glial fibrillary acidic protein and nestin, compared to neurospheres cultured in the presence of these factors. In this work, we show that rat neurospheres cultured in suspension under mitogen-free condition presented significant higher expression of P2X2 and P2X6 receptor subunits, when compared to cells cultured in the presence of growth factors, suggesting a direct relationship between P2X2/6 receptor expression and induction of neuronal differentiation in mitogen-free cultured rat neurospheres.

Adult CNS explants as a source of neural progenitors

Adult neural progenitors have been isolated from diverse regions of the CNS using methods which primarily involve the enzymatic digestion of tissue pieces; however, interpretation of these experiments can be complicated by the loss of anatomical resolution during the isolation procedures. We have developed a novel, explant-based technique for the isolation of neural progenitors, Living CNS regions were sectioned using a vibratome and small, well-defined discs of tissue punched out. When Cultured. explants from the cortex, hippocampus, cerebellum, spinal cord, hypothalamus, and caudate nucleus all robustly gave rise to proliferating progenitors. These progenitors were similar in behaviour and morphology to previously characterised multipotent hippocampal progenitor lines. Clones from all regions examined could proliferate from single cells and give rise to secondary neurospheres at a low but consistent frequency. Immunostaining demonstrated that clonal cortical progenitors were able to differentiate into both neurons and glial cells, indicating their multipotent characteristics. These results demonstrate it is possible to isolate anatomically resolved adult neural progenitors from small amounts of tissue throughout the CNS, thus, providing a tool for investigating the frequency and characteristics of progenitor cells from different regions. (c) 2005 Elsevier B.V. All rights reserved.

From neural stem cells to precursors : Molecular regulation of self-renewal and differentiation

Stem cells are responsible for tissue turnover throughout lifespan. Only highly controlled specific environment, the stem cell niche , can sustain undifferentiated stem cell-pool. The balance between maintenance and differentiation is crucial for individual s health: uncontrolled stem cell self-renewal or proliferation can lead to hyperplasia and mutations that further provoke malignant transformation of the cells. On the other hand, uninhibited differentiation may result in diminished stem cell population, which is unable to maintain tissue turnover. The mechanisms that control the switch from maintenance to differentiation in stem cells are not well known. The same mechanisms that direct the self-renewal and proliferation in normal stem cells are likely to be also involved in maintenance of cancer stem cell . Cancer stem cells exhibit stem cell like properties such as self-renewal- and differentiation capacity and they can also regenerate the tumor tissue. In this thesis, I have investigated the effect of classical oncogenes E6/E7 and c-Myc, tumor suppressors p53 and retinoblastoma (pRb) family, and vascular endothelial growth factor (VEGF) subfamily and glial cell line-derived neurothropic factor (GDNF) family ligands on behavior of embryonic neural stem cells (NSCs) and progenitors. The study includes also the characterization of cytoskeletal tumor suppressor neurofibromatosis 2 (NF2) protein merlin and ezrin-radixin-moesin (ERM) protein ezrin expression in neural progenitors cells and their progeny. This study reveals some potential mechanisms regarding to NSCs maintenance. In summary, the studied molecules are able to shift the balance either towards stem cell maintenance or differentiation; tumor suppressor p53 represses whereas E6/E7 oncogenes and c-Myc increase the proportion of self-renewing and proliferating NSCs or progenitors. The data suggests that active MEK-ERK signaling is critical for self-renewal of normal and oncogene expressing NSCs. In addition, the results indicate that expression of cytoskeletal tumor suppressor merlin and ERM protein ezrin in central nervous system (CNS) tissue and progenitors indicates their role in cell differentiation. Furthermore, the data suggests that VEGF-C a factor involved in lymphatic system development, angiogenesis, neovascularization and metastasis but also in maintenance of some neural populations in brain is a novel thropic factor for progenitors in early sympathetic nervous system (SNS). It seems that VEGF-C dose dependently through ERK-pathway supports the proliferation and survival of early sympathetic progenitor cells, and the effect is comparable to that of GDNF family ligands.

Repressor element 1 silencing transcription factor couples loss of pluripotency with neural induction and neural differentiation

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia-like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multi-potent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.

TRANSCRIPTIONAL AND POST-TRANSLATIONAL MECHANISMS CONTRIBUTE TO MAINTENANCE OF REST IN NEURAL TUMORS

The RE-1 silencing transcription factor (REST) is an important regulator of normal nervous system development. It negatively regulates neuronal lineage specification in neural progenitors by binding to its consensus RE-1 element(s) located in the regulatory region of its target neuronal differentiation genes. The developmentally coordinated down-regulation of REST mRNA and protein in neural progenitors triggers terminal neurogenesis. REST is overexpressed in pediatric neural tumors such as medulloblastoma and neuroblastoma and is associated with poor neuronal differentiation. High REST protein correlate with poor prognosis for patients with medulloblastoma, however similar studies have not been done with neuroblastoma patients. Mechanism(s) underlying elevated REST levels medulloblastoma and neuroblastoma are unclear, and is the focus of this thesis project. We discovered that transcriptional and post-translational mechanisms govern REST mis-regulation in medulloblastoma and neuroblastoma. In medulloblastoma, REST transcript is aberrantly elevated in a subset of patient samples. Using loss of function and gain of function experiments, we provide evidence that the Hairy Enhancer of Split (HES1) protein represses REST transcription in medulloblastoma cell lines, modulates the expression of neuronal differentiation genes, and alters the survival potential of these cells in vitro. We also show that REST directly represses its own expression in an auto-regulatory feedback loop. Interestingly, our studies identified a novel interaction between REST and HES1. We also observed their co-occupancy at the RE-1 sites, thereby suggesting potential for co-regulation of REST expression. Our pharmacological studies in neuroblastoma using retinoic acid revealed that REST levels are controlled by transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms are mediated by modulation of E3 ligase or REST, SCFβ-TRCP, and contribute to resistance of some cells to retinoic acid treatment.

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximate to -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca2+ concentration, which was dependent on extracellular Ca2+ and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na+ channels, low- and high-voltage-activated Ca2+ channels and delayed-rectifier K+ channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

肝细胞生长因子促进猕猴胚胎干细胞来源的神经前体细胞的增殖

肝细胞生长因子（HGF）是一个多效应因子，在神经系统中具有重要作用， 但是对于HGF在早期神经系统发育（特别是哺乳动物）中的具体作用还不明确， 这方面的研究还很少。我们早前的研究发现采用HGF和G5 supplement结合EB法 可诱导猕猴胚胎干细胞（rESCs）定向分化成高纯度（88.3± 8.1%）的可移植的 神经前体细胞，但是HGF在整个分化过程中的具体作用及HGF与其它因子的关 系还不清楚。 本研究改进先前研究体系，用单层培养诱导体系代替EB法诱导体系，用bFGF 代替G5 supplement。即采用单层培养的方式，分别用同时含bFGF和HGF、只含 HGF或bFGF以及两者都没有添加的成分确定的分化液诱导rESCs向神经细胞分 化。并检测HGF对rESCs来源的神经前体细胞的增殖速度的影响，旨在进一步研 究HGF在整个分化过程中的具体作用及HGF与bFGF的关系。主要结论如下：1） 采用单层培养法，同时添加HGF和bFGF可诱导rESCs在两周内定向分化为高纯度 （>85%）的神经前体细胞，从而建立了一种更为简单的诱导rESCs分化成神经细 胞的方法；2）不同分化条件下都得到了相似比例的神经前体细胞，表明外源性 的HGF在诱导rESC向神经前体细胞转变的过程中对于神经细胞命运的决定并不 起作用；3）HGF能有效地促进rESCs来源的神经前体细胞的增殖，并且与bFGF 具有协同作用。

肝细胞生长因子促进猕猴胚胎干细胞来源的神经前体细胞的增殖

肝细胞牛长因子(hepatoeyte growth factor,HGF)足一个多效应因子,在神经系统中具有重要作用.早前的研究发现采用HGF和G5 supplement结合EB(embryoid body)法可诱导猕猴胍胎干细胞(rhesus embryonic stem cells,rESCs)定向分化成高纯度的可移植的神经前体细胞(neural progenitors),但对于HGF在整个诱导分化过程中的具体作用及机制还不清楚.本研究改进先前研究体系,采用单层培养法,同时添加HGF和bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)诱导rESCs在两周内定向分化为高纯度[(81.66±4.37)%]的神经前体细胞,并且单独添加HGF或bFGF以及两者都没有添加的条件下也得到了相似比例的神经前体细胞,表明外源性的HGF在诱导rESCs向神经前体细胞转变的过程中对十神经细胞命运的决定并不起作用;进一步研究发现HGF能有效地促进神经前体细胞的增殖,并且与bFGF具有协同作用.总之,本研究建立了一种更为简单的诱导rESCs分化成神经细胞的方法,发现外源性的HGF在rESCs向神经前体细胞分化的过程中并没有神经诱导的作用,但能与bFGF协同作用促进rESCs来源的神经前体细胞的增殖.

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化,得到了高比例的神经前体细胞(97.5±0.83)%(P＜0.05).这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力.在长期的传代培养中发现,随着培养时间的延长,nestin阳性的神经前体细胞比例下降,同时发育能力也发生了变化.在传代培养的早期,神经前体细胞发育为神经元的比例很高,几乎没有胶质细胞分化出来.随着培养时间的延长,胶质细胞的比例逐渐上升.这与体内神经系统的发育过程非常相似.进一步研究发现具有bHLH (basic helix-loop-helix) 结构域的转录因子neurogenein2(Ngn2) 和Olig2可能在这一变化中起重要作用.因此,人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式,为在体外研究人的神经发育和再生医学奠定了基础.