A Subset of ES-Cell-Derived Neural Cells Marked by Gene Targeting

Xian, Hai-Qing; McNichols, Elizabeth; Clair, Andrew St.; Gottlieb, David

doi:10.1634/stemcells.21-1-41

Cited by 42 publications

(41 citation statements)

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“…P19 cells were maintained in an undifferentiated state in RPMI-1640 medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL) in an incubator under 5% CO 2 . CCE cells were maintained in DMEM (Gibco-BRL) supplemented with 15% fetal bovine serum (FBS) (Gibco-BRL), 100 U/mL penicillin (Gibco-BRL), 50 lg/mL streptomycin (Gibco-BRL), 0.5 lM 2-mercaptoethanol (2ME) and 500 pM LIF (StemCell Technologies, Vancouver, BC, Canada) (Xian et al 2003;Nishikawa et al 1998). The cells were cultured on 60 mm petri dishes (Nunc) coated with 1% gelatin (Sigma) in phosphate buffered saline, pH 7.2-7.4.…”

Section: Methodsmentioning

confidence: 99%

“…The cultures were kept for 48 h, after which the number of EBs was counted. RA (0.5 lM) (Xian et al 2003) was added for a further 2 days. The aggregated cells were transferred to plastic tissue culture grade dishes (Nunc) surface coated with 1% gelatin that contained DMEM supplemented with 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

“…Many investigators have shown that different chemicals, such as retinoic acid and neurogenic medium, can induce the differentiation of ESCs into neuroepithelial cells (Gottlieb and Huettner 1999;Brustle et al 1999;Chung et al 2002). ESCs should first be allowed to form embryoid bodies (EBs) in culture media without inhibitory factors of differentiation, such as leukemia inhibitory factor (Yamada et al 2002), STO cell line (Xian et al 2003), and primary embryo fibroblast (Nishikawa et al 1998). There are several techniques to encourage EB formation, which include the suspended drop method, the bacteriological culture petri dish method, and using semisolid substrates, such as methylcellulose.…”

Section: Introductionmentioning

confidence: 99%

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Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

et al. 2009

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Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote TM as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote TM , as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the siliconcoating of glass petri dishes by Sigmacote TM is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

et al. 2009

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“…À ce niveau, les progéni-teurs semblent incapables de donner naissance à des populations oligodendrocytaires, suggérant une origine distincte de celle des progéniteurs oligo-astrocytaires ventraux. Dans certaines conditions de culture, les cellules ES se différen-cient en cellules gliales [41,43]. Les corps embryoïdes peuvent produire des cellules astrogliales, oligodendrogliales et microgliales (visualisées respectivement par les marqueurs GFAP, O4 et 5C6 ou galectin-3) selon un processus séquentiel qui rappelle la dynamique des mécanismes mis en jeu in utero [44].…”

Section: Différenciation Gliale (Astrocytes Et Oligodendrocytes)unclassified

Différenciation neurale des cellules souches embryonnaires

2005

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Les cellules souches embryonnaires murines ES sont isolées directement en culture à partir de la masse cellulaire interne (ICM) d'embryons âgés de 3,5 jours (blastocystes) [1,2]. Elles sont pluripotentes et capables de se différencier in vivo dans tous les organes de l'embryon et, in vitro, dans la plupart des types cellulaires, à l'exception des tissus extra-embryonnaires. Les résul-tats actuellement disponibles indiquent que les cellules ES se différencient en suivant un programme génétique comparable (en termes de molécules, voies de signalisation, étapes et chronologie) à celui des cellules souches neurales au cours du développement embryonnaire. Expérimentale-ment, les cellules ES possèdent un potentiel dans les domaines de la thérapie cardiaque, pulmonaire, hépatique ou encore de la régénération osseuse et articulaire [3], mais c'est dans le traitement des maladies cérébrales dégénéra-tives que la technique ouvre ses perspectives les plus intéressantes [4,5]. Dans cet article, nous nous focaliserons sur la différenciation neurale des cellules souches embryonnaires murines ES, en suivant l'idée selon laquelle les cellules ES se différencient in vitro selon un programme génétique similaire à celui qui régit le développement embryonnaire in utero. Aspects techniques de la différenciation des cellules ESDeux méthodes principales sont utilisées pour différen-cier les cellules ES en cellules neurales. La première fait appel à la formation des corps embryoïdes, qui déve-loppent les trois feuillets germinatifs. Ils représentent un succédané, in vitro, des embryons au stade péri-implantatoire: les cellules produisent une population mixte contenant en proportion variable des neurones et des cellules gliales (astrocytes et oligodendrocytes). La seconde méthode consiste à cultiver les cellules ES en milieu sans sérum, conditionné ou non par différents morphogènes : les cellules ES produisent alors des > Les cellules souches embryonnaires pluripotentes (ES) ont des propriétés de prolifération et de différenciation qui font d'elles un outil prometteur pour la thérapie cellulaire. Malgré la légitime controverse sur les nombreuses questions éthiques que cette technologie suscite, et en dépit de ses potentiels dans des domaines aussi variables que la thérapie cardiaque, pulmonaire ou hépatique, ou la régénération osseuse et articulaire, c'est dans le traitement des maladies cérébrales que la technique ouvre les perspectives les plus intéressantes. De très nombreux travaux réalisés sur les cellules ES rapportent une régénération du cerveau et de la moelle épinière chez des rongeurs et la répara-tion des problèmes cognitifs causés lors de dommages apportés aux tissus lésés. Cet article offre une synthèse des avancées récentes dans le domaine de la manipulation des cellules souches (murines) en vue de sélectionner des populations de neurones, astrocytes et oligodendrocytes. En parallèle, nous mettons l'accent sur les similitudes frappantes qui existent dans les programmes génétiques mis en oeuvre au cours du développement emb...

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“…The mESC culture medium is Dulbecco's modified Eagle's medium (DMEM) (GIBCO) supplemented with 20% fetal bovine serum (GIBCO), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol/, 1% nonessential amino acids (NEAA), and 1,000 U/ml leukemia inhibitory factor. 2. mES colonies are trypsinized (TrypLE, 5 min, 37°C) into single cells and suspended in knockout serum replacement (KSR) medium, then the cells are transferred to a Costar ultra-low attachment six-well plate (Corning) at a cell density of 50,000 cells/cm 2 . In these conditions, mESCs form embryoid bodies (EBs).…”

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confidence: 99%

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Jiang¹,

Selvaraj²,

Deng³

2010

JoVE

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Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (>80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing Na V 1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

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A Subset of ES-Cell-Derived Neural Cells Marked by Gene Targeting

Abstract: ABSTRACT

Cited by 42 publications

References 24 publications

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Formation of embryoid bodies from mouse embryonic stem cells cultured on silicon-coated surfaces

Différenciation neurale des cellules souches embryonnaires

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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