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In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice,while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover,gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice,resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells,and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline. View Publication

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells,including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells,known as brain tumor initiating cells,likely contribute to glioma recurrence,as they are highly invasive,mobile,resistant to radiation and chemotherapy,and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate,which may be a key process in the death of peritumoral neurons,formation of necrosis,local inflammation,and glioma-related seizures. Moreover,elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells,resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells,which express the stem cell markers nestin and SOX-2,we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors,compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting,immunocytochemistry,and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However,such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs),which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods,we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene,which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication

For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies. View Publication

The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly,however,studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here,we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly,however,depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (textgreater 250 microm in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast,the same conditions led to the opposite effect in the other main neurogenic region of the brain,the subventricular zone,in which neurosphere numbers decreased by approximately 40% in response to depolarizing levels of KCl. Most importantly,we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus,which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal,defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis. View Publication