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Showing papers on "Neurosphere published in 2009"


Journal ArticleDOI
TL;DR: Routine and efficient derivation of adherent cell lines from malignant glioma that display stem cell properties and initiate high-grade gliomas following xenotransplantation are demonstrated.

946 citations


Journal ArticleDOI
TL;DR: This study evaluated the teratoma-forming propensity of secondary neurospheres generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways, which correlated with the persistence of undifferentiated cells.
Abstract: We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast-derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells.

884 citations


Journal ArticleDOI
TL;DR: In normal brain, the side population (SP) phenotype is generated by ABC transporter activity and identifies stem cell and endothelial cell subpopulations by dye exclusion, and is increased in nonendothelial cells that form neurospheres and are highly tumorigenic.

756 citations


Journal ArticleDOI
01 Oct 2009-Nature
TL;DR: It is demonstrated that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency and will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.
Abstract: Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

737 citations


Journal ArticleDOI
TL;DR: It is found that hypoxia promotes the self-renewal capability of the stem and non- stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation and upregulation of important stem cell factors, such as OCT4, NANOG, and c-MYC.
Abstract: Glioblastomas are highly lethal cancers that contain cellular hierarchies with self-renewing cancer stem cells that can propagate tumors in secondary transplant assays. The potential significance of cancer stem cells in cancer biology has been demonstrated by studies showing contributions to therapeutic resistance, angiogenesis and tumor dispersal. We recently reported that physiologic oxygen levels differentially induce hypoxia inducible factor-2alpha (HIF2alpha) levels in cancer stem cells. HIF1alpha functioned in proliferation and survival of all cancer cells but also was activated in normal neural progenitors suggesting a potentially restricted therapeutic index while HIF2alpha was essential in only in cancer stem cells and was not expressed by normal neural progenitors demonstrating HIF2alpha is a cancer stem cell specific target. We now extend these studies to examine the role of hypoxia in regulating tumor cell plasticity. We find that hypoxia promotes the self-renewal capability of the stem and non-stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation as well as upregulation of important stem cell factors, such as OCT4, NANOG and c-MYC. The importance of HIF2alpha was further supported as forced expression of non-degradable HIF2alpha induced a cancer stem cell marker and augmented the tumorigenic potential of the non-stem population. This novel finding may indicate a specific role of HIF2alpha in promoting glioma tumorigenesis. The unexpected plasticity of the non-stem glioma population and the stem-like phenotype emphasizes the importance of developing therapeutic strategies targeting the microenvironmental influence on the tumor in addition to cancer stem cells.

714 citations


Journal ArticleDOI
TL;DR: Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.
Abstract: An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.

501 citations


Journal ArticleDOI
15 Aug 2009-Glia
TL;DR: It is demonstrated that human bone marrow‐derived MSCs (BM‐hMSCs) promote functional recovery in both chronic and relapsing‐remitting models of mouse EAE, and their ability to modulate disease progression and the host immune response is tested.
Abstract: Cell based therapies are attractive approaches to promote myelin repair. Recent studies demonstrated a reduction in disease burden in mice with EAE treated with mouse mesenchymal stem cells (MSCs). Here we demonstrated human bone marrow derived MSCs (BM-hMSCs) promote functional recovery in both chronic and relapsing-remitting models of mouse EAE, traced their migration into the injured CNS and assayed their ability to modulate disease progression and the host immune response. Injected BM-hMSCs accumulated in the CNS, reduced the extent of damage and increased oligodendrocyte lineage cells in lesion areas. The increase in oligodendrocytes in lesions may reflect BM-hMSC induced changes in neural fate determination since neurospheres from treated animals gave rise to more oligodendrocytes and less astrocytes than non-treated neurospheres. Host immune responses were also influenced by BM-hMSCs. Inflammatory T-cells including interferon gamma (IFN-γ) producing Th1 cells and IL-17 producing Th17 inflammatory cells and their associated cytokines were reduced along with concomitant increases in IL-4 producing Th2 cells and anti-inflammatory cytokines. Together these data suggest the BM-hMSCs represent a viable option for therapeutic approaches.

486 citations


Journal ArticleDOI
TL;DR: This study examines the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth.

439 citations


Journal ArticleDOI
TL;DR: Treatment of putative glioblastoma stem cells with two chemically distinct small molecule inhibitors of STAT3 DNA‐binding inhibits cell proliferation and the formation of new neurospheres from single cells, suggesting that STAT3 regulates the growth and self‐renewal of GBM‐SCs and is thus a potential target for cancer stem cell‐directed therapy of gliOBlastoma multiforme.
Abstract: Signal transducer and activator of transcription 3 (STAT3) regulates diverse cellular processes, including cell growth, differentiation, and apoptosis, and is frequently activated during tumorigenesis. Recently, putative glioblastoma stem cells (GBM-SCs) were isolated and characterized. These cells can self-renew indefinitely in culture, are highly tumorigenic, and retain the ability to differentiate in culture. We have found that treatment of GBM-SCs with two chemically distinct small molecule inhibitors of STAT3 DNA-binding inhibits cell proliferation and the formation of new neurospheres from single cells. Genetic knockdown of STAT3 using a short hairpin RNA also inhibits GBM-SC proliferation and neurosphere formation, confirming that these effects are specific to STAT3. Although STAT3 inhibition can induce apoptosis in serum-derived GBM cell lines, this effect was not observed in GBM-SCs grown in stem cell medium. Markers of neural stem cell multipotency also decrease upon STAT3 inhibition, suggesting that STAT3 is required for maintenance of the stem-like characteristics of these cells. Strikingly, even a transient inhibition of STAT3 leads to irreversible growth arrest and inhibition of neurosphere formation. These data suggest that STAT3 regulates the growth and self-renewal of GBM-SCs and is thus a potential target for cancer stem cell-directed therapy of glioblastoma multiforme.

429 citations


Journal ArticleDOI
TL;DR: It is shown that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment.
Abstract: The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP(+)EGFR(+)CD24(-)), niche astrocytes (GFP(+)EGFR(-)CD24(-)), transit amplifying cells (GFP(-)EGFR(+)CD24(-)), and neuroblasts (GFP(-)EGFR(-)CD24(low)). One in three EGFR(+) astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphere-forming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.

365 citations


Journal ArticleDOI
TL;DR: The relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-Renewal.
Abstract: Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal.

Journal ArticleDOI
TL;DR: These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSVs mutations, important for designing new o HSV vectors and clinical trials.
Abstract: Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem–like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)–deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of γ34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of α47. Infection with oHSV G47Δ (ICP6−, γ34.5−, α47−) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Δ significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis. [Cancer Res 2009;69(8):3472–81]

Journal ArticleDOI
TL;DR: Results indicate that the induction of autophagy contributes to the radioresistance of these cells and Autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to γ‐radiation.
Abstract: Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo- and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to gamma-radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. gamma-radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by gamma-radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24-48 hr and slightly decreased the viability of the cells. gamma-radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133- cells and the CD133+ cells expressed higher levels of the autophagy-related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to gamma-radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to gamma-radiation.

Journal ArticleDOI
TL;DR: The data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for gliomas patients.
Abstract: Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.

Journal ArticleDOI
TL;DR: It is suggested that high expression of a single transcriptional regulator, Id1, molecularly defines the long-sought-after B1 type adult neural stem cells.

Journal ArticleDOI
TL;DR: GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro and p53 regulates the phosphorylation of a novel N’ truncated putative activator isoform of GLI1 in human cells.
Abstract: How cell numbers are determined is not understood. Hedgehog-Gli activity is involved in precursor cell proliferation and stem cell self-renewal, and its deregulation sustains the growth of many human tumours. However, it is not known whether GLI1, the final mediator of Hh signals, controls stem cell numbers, and how its activity is restricted to curtail tumourigenesis. Here we have altered the levels of GLI1 and p53, the major tumour suppressor, in multiple systems. We show that GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro. In contrast, p53 inhibits GLI1-driven neural stem cell self-renewal, tumour growth and proliferation. Mechanistically, p53 inhibits the activity, nuclear localisation and levels of GLI1 and in turn, GLI1 represses p53, establishing an inhibitory loop. We also find that p53 regulates the phosphorylation of a novel N' truncated putative activator isoform of GLI1 in human cells. The balance of GLI1 and p53 functions, thus, determines cell numbers, and prevalence of p53 restricts GLI1-driven stem cell expansion and tumourigenesis.

Journal ArticleDOI
TL;DR: The results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.

Journal ArticleDOI
TL;DR: Growth at the more physiologically relevant oxygen level of 7% enhances the stem cell–like phenotype of CD133+ GB cells, andGene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs.
Abstract: In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

Journal ArticleDOI
TL;DR: It is found that expression of Hes1 and its downstream genes oscillate in mouse embryonic stem cells, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells even under the same environmental conditions.
Abstract: Stem cells do not all respond the same way, but the mechanisms underlying this heterogeneity are not well understood. Here, we found that expression of Hes1 and its downstream genes oscillate in mouse embryonic stem (ES) cells. Those expressing low and high levels of Hes1 tended to differentiate into neural and mesodermal cells, respectively. Furthermore, inactivation of Hes1 facilitated neural differentiation more uniformly at earlier time. Thus, Hes1-null ES cells display less heterogeneity in both the differentiation timing and fate choice, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells even under the same environmental conditions.

Journal ArticleDOI
21 Jul 2009-PLOS ONE
TL;DR: This work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.
Abstract: Background The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. Conclusions/Significance Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

Journal ArticleDOI
TL;DR: Results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma and suggest that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome.
Abstract: Renewable neurosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. This retrospective study was designed to assess the relationship among neurosphere formation in cultured human glioma, tumorigenic capacity, and patient clinical outcome. Tumor samples were cultured in neurosphere conditions from 32 patients with glioma, including a subpopulation of 15 patients with primary glioblastoma. A subsample of renewable neurosphere cultures was xenografted into mouse brain to determine if they were tumorigenic. Our study shows that both renewable neurosphere formation and tumorigenic capacity are significantly associated with clinical outcome measures. Renewable neurosphere formation in cultured human glioma significantly predicted an increased hazard of patient death and more rapid tumor progression. These results pertained to both the full population of glioma and the subpopulation of primary glioblastoma. Similarly, there was a significant hazard of progression for patients whose glioma had tumorigenic capacity. Multivariate analysis demonstrated that neurosphere formation remained a significant predictor of clinical outcome independent of Ki67 proliferation index. In addition, multivariate analysis of neurosphere formation, tumor grade and patient age, demonstrated that neurosphere formation was a robust, independent predictor of glioma tumor progression. Although the lengthy duration of this assay may preclude direct clinical application, these results exemplify how neurosphere culture serves as a clinically relevant model for the study of malignant glioma. Furthermore, this study suggests that the ability to propagate brain tumor stem cells in vitro is associated with clinical outcome.

Journal ArticleDOI
TL;DR: In this paper, the authors identify a CD surface antigen code for the neural lineage based on combinatorial flow cytometric analysis of three distinct populations derived from human embryonic stem cells.
Abstract: Identification and use of cell surface cluster of differentiation (CD) biomarkers have enabled much scientific and clinical progress. We identify a CD surface antigen code for the neural lineage based on combinatorial flow cytometric analysis of three distinct populations derived from human embryonic stem cells: (1) CD15+/CD29HI/CD24LO surface antigen expression defined neural stem cells; (2) CD15−/CD29HI/CD24LO revealed neural crest-like and mesenchymal phenotypes; and (3) CD15−/CD29LO/CD24HI selected neuroblasts and neurons. Fluorescence-activated cell sorting (FACS) for the CD15−/CD29LO/CD24HI profile reduced proliferative cell types in human embryonic stem cell differentiation. This eliminated tumor formation in vivo, resulting in pure neuronal grafts. In conclusion, combinatorial CD15/CD24/CD29 marker profiles define neural lineage development of neural stem cell, neural crest, and neuronal populations from human stem cells. We believe this set of biomarkers enables analysis and selection of neural cell types for developmental studies and pharmacological and therapeutic applications. STEM CELLS 2009;27:2928–2940

Journal ArticleDOI
TL;DR: It is reported here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells, and provides an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitors cell.
Abstract: It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Journal ArticleDOI
11 Jun 2009-Oncogene
TL;DR: It is shown that the cell of origin for glioma may be a committed glial progenitor cell, and platelet-derived growth factor B transfer to OPCs could induce gliomas with an incidence of 33%.
Abstract: Gliomas are primary brain tumors mainly affecting adults. The cellular origin is unknown. The recent identification of tumor-initiating cells in glioma, which share many similarities with normal neural stem cells, has suggested the cell of origin to be a transformed neural stem cell. In previous studies, using the RCAS/tv-a mouse model, platelet-derived growth factor B (PDGF-B)-induced gliomas have been generated from nestin or glial fibrillary acidic protein-expressing cells, markers of neural stem cells. To investigate if committed glial progenitor cells could be the cell of origin for glioma, we generated the Ctv-a mouse where tumor induction would be restricted to myelinating oligodendrocyte progenitor cells (OPCs) expressing 2',3'-cyclic nucleotide 3'-phosphodiesterase. We showed that PDGF-B transfer to OPCs could induce gliomas with an incidence of 33%. The majority of tumors resembled human WHO grade II oligodendroglioma based on close similarities in histopathology and expression of cellular markers. Thus, with the Ctv-a mouse we have showed that the cell of origin for glioma may be a committed glial progenitor cell.

Journal ArticleDOI
TL;DR: It is shown that soft substrates with elasticities commensurable to the elasticity of the brain promote the maturation of neural stem cell-derived neurons, defining a crucial parameter in neuronal differentiation of stem cells.

Journal ArticleDOI
TL;DR: It is shown that bipotent stem cells are maintained in a distinct niche in the adult zebrafish cerebellum and retain neuroepithelial characteristics, which are critical common determinants to maintain neural stem cell activity in vertebrates.
Abstract: In the adult CNS, neurogenesis takes place in special niches. It is not understood how these niches are formed during development and how they are maintained. In contrast to mammals, stem cell niches are abundant in zebrafish and also found in other parts of the brain than telencephalon. To understand common characteristics of neural stem cell niches in vertebrates, we studied the origin and architecture of a previously unknown stem cell niche using transgenic lines, in vivo imaging, and marker analysis. We show that bipotent stem cells are maintained in a distinct niche in the adult zebrafish cerebellum. Remarkably, the stem cells are not typical glia but instead retain neuroepithelial characteristics. The cerebellar stem cell niche is generated by the coordinated displacement of ventricle and rhombic lip progenitors in a two-step process involving morphogenetic movements and tissue growth. Importantly, the niche and its stem cells still remain in ventricular contact through a previously unknown derivative of the ventricle. Factors propagated in the ventricle are thought to be important regulators of stem cell activity. To test the requirements of one family of important factors, Fibroblast growth factors, we used zebrafish with an inducible dominant-negative Fgf receptor. Inhibition of Fgf signaling leads to significant reduction of stem cell activity. In contrast to the predominant view, adult neural stem cells in nonmammalian vertebrates show more neuroepithelial than glial characteristics. Nevertheless, retained epithelial properties such as distinct polarization and ventricular contact are critical common determinants to maintain neural stem cell activity in vertebrates.

Journal ArticleDOI
TL;DR: It is suggested that glioblastoma stem like cells were existed in CD133+ cells in U251 cell line with characteristics of self-renew and generation of an unlimited progeny of non-tumorigenic cells.

Journal ArticleDOI
TL;DR: It is demonstrated that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133+ and CD133− GBM BTSCs from neural stem cells.
Abstract: Primary glial tumors of the central nervous system, most commonly glioblastoma multiforme (GBM), are aggressive lesions with a dismal prognosis. Despite identification and isolation of human brain tumor stem cells (BTSCs), characteristics that distinguish BTSCs from neural stem cells remain to be elucidated. We cultured cells isolated from gliomas, using the neurosphere culture system, to understand their growth requirements. Both CD133(+) and CD133(-) adult GBM BTSCs proliferated in the absence of exogenous mitogenic stimulation and gave rise to multipotent GBM spheres that were capable of self-renewal. Epidermal growth factor (EGF) and fibroblast growth factor-2 enhanced GBM BTSC survival, proliferation, and subsequent sphere size. Blockade of EGF receptor (EGFR) signaling reduced exogenous mitogen-independent GBM sphere growth. Implantation of as few as 10 exogenous mitogen-independent GBM BTSCs led to the formation of highly invasive intracranial tumors, which closely resembled human GBMs, in immunocompromised mice. These results demonstrate that exogenous mitogen independence, mediated in part through EGFR signaling, is one characteristic that distinguishes CD133(+) and CD133(-) GBM BTSCs from neural stem cells. This novel experimental system will permit the elucidation of additional constitutively activated mechanisms that promote GBM BTSC survival, self-renewal, and proliferation.

Journal ArticleDOI
TL;DR: DCAMKL‐1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR5+), which represents a novel research tool for regenerative medicine and cancer therapy.
Abstract: It is thought that small intestinal epithelia (IE) undergo continuous self-renewal primarily due to their population of undifferentiated stem cells. These stem cells give rise to transit amplifying (daughter/progenitor) cells, which can differentiate into all mature cell types required for normal gut function. Identification of stem cells in IE is paramount to fully understanding this renewal process. One major obstacle in gastrointestinal stem cell biology has been the lack of definitive markers that identify small intestinal stem cells (ISCs). Here we demonstrate that the novel putative ISC marker doublecortin and CaM kinase-like-1 (DCAMKL-1) is predominantly expressed in quiescent cells in the lower two-thirds of intestinal crypt epithelium and in occasional crypt-based columnar cells (CBCs). In contrast, the novel putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor (LGR5) is observed in rapidly cycling CBCs and in occasional crypt epithelial cells. Furthermore, functionally quiescent DCAMKL-1+ crypt epithelial cells retain bromo-deoxyuridine in a modified label retention assay. Moreover, we demonstrate that DCAMKL-1 is a cell surface expressing protein; DCAMKL-1+ cells, isolated from the adult mouse small intestine by fluorescence activated cell sorting, self-renew and ultimately form spheroids in suspension culture. These spheroids formed glandular epithelial structures in the flanks of athymic nude mice, which expressed multiple markers of gut epithelial lineage. Thus, DCAMKL-1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR51). Moreover, DCAMKL-1 can be used to isolate normal small intestinal stem cells and represents a novel research tool for regenerative medicine and cancer therapy.

Journal ArticleDOI
TL;DR: In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord.
Abstract: Biodegradable polymer scaffolds provide an excellent approach to quantifying critical factors necessary for restoration of function after a transection spinal cord injury. Neural stem cells (NSCs) and Schwann cells (SCs) support axonal regeneration. This study examines the compatibility of NSCs and SCs with the poly-lactic-co-glycolic acid polymer scaffold and quantitatively assesses their potential to promote regeneration after a spinal cord transection injury in rats. NSCs were cultured as neurospheres and characterized by immunostaining for nestin (NSCs), glial fibrillary acidic protein (GFAP) (astrocytes), betaIII-tubulin (immature neurons), oligodendrocyte-4 (immature oligodendrocytes), and myelin oligodendrocyte (mature oligodendrocytes), while SCs were characterized by immunostaining for S-100. Rats with transection injuries received scaffold implants containing NSCs (n=17), SCs (n=17), and no cells (control) (n=8). The degree of axonal regeneration was determined by counting neurofilament-stained axons through the scaffold channels 1 month after transplantation. Serial sectioning through the scaffold channels in NSC- and SC-treated groups revealed the presence of nestin, neurofilament, S-100, and betaIII tubulin-positive cells. GFAP-positive cells were only seen at the spinal cord-scaffold border. There were significantly more axons in the NSC- and SC- treated groups compared to the control group. In conclusion, biodegradable scaffolds with aligned columns seeded with NSCs or SCs facilitate regeneration across the transected spinal cord. Further, these multichannel biodegradable polymer scaffolds effectively serve as platforms for quantitative analysis of axonal regeneration.