Ce travail a été en partie soutenu par des subventions à partir de DEO National Institutes of Health (RO1 NS059043 et RO1 ES015988), National Multiple Sclerosis Society, Roche Foundation for recherche de l&#39;anémie, Feldstein Fondation médicale, et les Hôpitaux Shriners pour enfants. Nous tenons à remercier le Dr David Plaisir et Jennifer Avion pour des suggestions. Nous déclarons sans intérêts concurrents liés à cet article.

Les procédures détaillées de générer des cellules précurseurs des oligodendrocytes (OPC) de cellules de souris GFP-Olig2 souches embryonnaires (mESCs): <ol><li> Ligne de cellules ES de souris GFP-Olig2 (G-Olig2), est acheté à l&#39;American Type Culture Collection (ATCC). Le mESCs sont régulièrement repiquées tous les 3 jours sur un embryon de souris irradiées couche de desserte des fibroblastes (MEF). Les cellules MEF sont étalées dans 0,1% de gélatine enduit de six puits plaque de culture tissulaire au moins un jour avant le repiquage du CES. Le milieu de culture MESC est moyen Eagle modifié par Dulbecco (DMEM) (Gibco) supplémenté avec 20% sérum de veau fœtal (Gibco), 2 mM L-glutamine, 1 mM pyruvate de sodium, 0,1 mM β-mercaptoéthanol /, 1% acides aminés non essentiels ( NEAA), et 1000 U / ml de facteur inhibiteur de leucémie. </li><li> MES colonies sont trypsinisées (TrypLE, 5 min, 37 ° C) dans des cellules individuelles et suspendu en huitièmes de finale de remplacement (KSR) milieu sans sérum, puis les cellules sont transférées à un Costar ultra-faible attachement à six puits plaque (Corning) à une cellule la densité de 50.000 cellules / cm 2. Dans ces conditions, mESCs forment des corps embryoïdes (EBS). Moyenne se compose de KSR-milieu essentiel minimal (MEM-) complété avec KSR 20%, 1 mM pyruvate de sodium, NEAA 1%, et 0,1 mM β-mercaptoéthanol /. </li><li> Du jour 4 au jour 7, l&#39;acide rétinoïque (RA, 0,2 M) et purmorphamine (Pur, 1 M) sont inclus, comme indiqué dans la Fig. 1A dans KSR ou moyen N2. Le milieu N2 est-MEM contenant complément N2 1X, 1 mM pyruvate de sodium, NEAA 1%, et 0,1 mM β-mercaptoéthanol /. Le milieu est changé tous les jours. </li><li> Au jour 8, EBS sont ventilées en utilisant TrypLE (5min, 37 ° C) et plaqué sur 0,01% polyornithine enduits plats en milieu OPC qui se compose de moyennes et de croissance des fibroblastes N2 facteur-2 (FGF-2, 20 ng / ml). Le milieu est changé tous les deux jours. </li><li> Les cellules sont trypsinisées (TrypLE, 3 min, 37 ° C) et réétalées environ chaque semaine quand ils deviennent confluentes. A 30 jours, plus de 80% des cellules montrent GFP/Olig2 expression et sont NG2-positif, caractéristique des OPC. </li><li> Pour générer les OPC de dopage, le Na + canal BacMam kit est utilisé pour introduire Na v 1.2 sous-unité dans ces OPC MESC dérivés. Le réactif BacMam (1 ml, composant A) est mélangé avec du PBS pour un volume final de 5 ml. Ensuite, le réactif BacMam mixte est ajouté dans MESC dérivées de la culture OPC dans une boîte de culture de 60 mm (à 50-70% de confluence) avant le rinçage avec du PBS. Après une incubation de 2 h, le réactif BacMam mixte est supprimé et remplacé par du milieu OPC complétée par 1X Enhancer. L&#39;activateur est le composant B reconstitué dans du DMSO (composante C). Ensuite, après 2 heures supplémentaires d&#39;incubation du milieu avec Enhancer est retiré et remplacé par du milieu complet OPC. Les cellules sont analysés 24 heures plus tard. </li></ol> Les résultats représentatifs: Après le protocole de différenciation Fig. 1A, G-Olig2 mESCs avaient été suspendus dans le milieu de KSR et pendant 4 jours pour former des corps embryoïdes (EB) (Fig. 1B). Ensuite, nous avons traité de façon séquentielle l&#39;EBS avec AR et Pur. L&#39;EBS n&#39;a pas montré de fluorescence de la GFP au jour 4 et ont exprimé une forte fluorescence de la GFP à J8 (Fig. 1B). A ce point du temps, l&#39;EBS ont été trypsinées et plaqués en milieu N2 complété avec des facteurs de croissance FGF-2. Après une nouvelle culture pour 22 jours (J30), le pourcentage de cellules GFP + a atteint 80,3 ± 0,6%, selon nos résultats de cytométrie en flux. Comme le montre la Fig. 1C, cellules GFP + double emploi avec NG2 coloration constante (96,4 ± 1,3% de cellules GFP-positives ont été NG2 positif, et 94,8 ± 1,5% de cellules positives ont été NG2 GFP-positives). Ainsi, ces cellules expriment GFP/Olig2 et NG2, les définissant comme les OPC. L&#39;OPC MESC dérivés (76 cellules) a échoué au feu des potentiels d&#39;action sur la dépolarisation (figure 2A). Après transduction avec un baculovirus portant le Na v 1.2 sous-unité, ces OPC MESC dérivés (94,1%, 16 des 17 cellules) pourraient être classés comme enrichissement (figure 2B). <img alt="Figure 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Figure 1. Différenciation des GOlig-MESC dans les OPC. (A) Schéma montrant le protocole du corps embryoïdes (EB) à base de petites molécules et axée sur la différenciation. A J8, l&#39;EBS ont été ventilées et plaqué. Les cellules ont été repiquées une fois par semaine quand ils sont devenus confluentes. (B) D8, mais pas D4 EBS, a montré expression de la GFP. (C) Immunocoloration des NG2 (rouge) est compatible avec la GFP (green) l&#39;expression à J30. DAPI (bleu) a été utilisé pour identifier les noyaux. Barre d&#39;échelle: 20 mm. <img alt="Figure 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Figure 2. Génération d&#39;OPC de dopage à partir nonspiking MESC dérivé OPC par le virus médiée par le canal Na v expression. (A) Le potentiel de membrane a été tenue à -60 mV et MESC dérivé OPC échoué au feu des potentiels d&#39;action, même lorsque la cellule est dépolarisée à 0 mV. (B) Un exemple de MESC dérivé OPCtir potentiel d&#39;action (en rouge) après la transduction virale.

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

Les cellules souches embryonnaires (CSE), isolé à partir d&#39;un embryon de blastocyste, peuvent se différencier en tous les lignées cellulaires de l&#39;organisme 3, 4, fournissant un système modèle in vitro pour étudier le développement précoce des mammifères, y compris la spécification des oligodendrocytes. mESCs a été démontré que de se différencier en cellules précurseurs des oligodendrocytes (OPC) avec le traitement de Sonic Hedgehog (Shh) 5. Par ailleurs, la différenciation induite par Shh OPC de mESCs conserve la synchronisation correcte observés dans le développement embryonnaire 6. Ainsi, la nature du diagnostic in vitro OPC différenciation par mESCs est considéré comme compatible avec ce qui a été appris de développement in vivo. Ici, en utilisant les petites molécules et la RA Pur agoniste Shh, nous avons réussi à différencier l&#39;mESCs G-Olig2 en GFP + + Olig2 NG2 + OPC avec une grande efficacité. OPC, caractérisé par l&#39;expression du protéoglycane NG2 7 et le facteur de transcription hélice-boucle-hélice Olig2 8, générer des oligodendrocytes dans le SNC en développement et matures, où ils représentent une proportion importante (~ 5%) du nombre total de cellules et sont les principaux types de cellules proliférantes 9. Pour les chercheurs de près de deux décennies ont démontré Na canaux V sont exprimés dans une sous-population d&#39;OPC et peut être activé à la dépolarisation 10, 11. Par ailleurs, une récente observation frappante est que, dans neuf dans le système nerveux central chez le rat in situ de la matière blanche, les OPC (~ 50%) ont généré des potentiels d&#39;action lors dépolarisée selon l&#39;expression de voltage-dépendants de sodium (Na V) des canaux, et pourrait donc être subdivisé en de dopage et nonspiking sous-populations. Cependant, les fonctions de ces propriétés de dopage sont encore largement inconnus. Nous avons constaté que, électrophysiologique, l&#39;OPC MESC dérivés différenciés avec le protocole Shh-dépendants, ne sont pas les mêmes que l&#39;OPC dans le cerveau in situ. Après l&#39;introduction de la sous-unité Na V 1.2, ces silencieux MESC dérivé OPC étaient capables de dopage. Ainsi, le dopage / nonspiking MESC dérivé OPC et le protocole de différenciation décrites ici peuvent faciliter (1) étudier les différences fonctionnelles entre dopage et nonspiking OPC, (2) les facteurs de dépistage de nouvelles qui pourraient favoriser l&#39;expression des canaux sodiques dans les OPCs MESC dérivés, ( 3) le développement et l&#39;optimisation du protocole de différenciation des OPC de CSE humaines ou des cellules souches pluripotentes induites (CISP).

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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
<td>&nbsp;</td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
<td>&nbsp;</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
<td>&nbsp;</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
<td>&nbsp;</td>
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<td>FGF-2</td>
<td>&nbsp;</td>
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<td>BacMam NaV 1.2</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

Les oligodendrocytes sont les cellules myélinisantes du système nerveux central. Pour la thérapie cellulaire régénérative dans les maladies démyélinisantes, il ya un intérêt significatif en dérivant une population pure de la lignée de cellules précurseurs commis-oligodendrocytes (OPC) pour la transplantation. Les OPC sont caractérisées par l&#39;activité du facteur de transcription Olig2 et d&#39;expression de surface d&#39;un NG2 protéoglycanes. Utilisation de la GFP-Olig2 (G-Olig2) de cellules souches embryonnaires de souris (MESC) ligne journaliste, nous avons optimisé les conditions de la différenciation des mESCs en GFP + + Olig2 NG2 + OPC. Dans notre protocole, nous décrivons d&#39;abord la génération des corps embryoïdes (EB) à partir mESCs. Deuxièmement, nous décrivons le traitement des MESC dérivés EBS avec de petites molécules: (1) l&#39;acide rétinoïque (AR) et (2) purmorphamine un hérisson Sonic agoniste (Shh) (Pur), dans des conditions de culture définie pour diriger une différenciation EB dans la lignée oligodendrocyte. Par cette approche, les OPC peuvent être obtenus avec un rendement élevé (&gt; 80%) dans un délai de 30 jours. Les cellules dérivées de mESCs dans ce protocole sont phénotypiquement similaire à OPCs issus de culture de tissus primaires. L&#39;OPC MESC dérivés ne montrent pas la propriété de dopage décrites pour une sous-population des OPC du cerveau in situ. Pour étudier cette propriété électrophysiologique, nous décrivons la génération de dopage MESC dérivé OPC par ectopique exprimer Na<sub&gt; V</sub&gt; 1,2 sous-unité. Les cellules de fortification et nonspiking obtenus à partir de ce protocole aidera les études fonctionnelles d&#39;avance sur les deux sous-populations d&#39;OPC.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Nous décrivons une molécule à base de petites protocole pour la différenciation des cellules souches embryonnaires de souris en cellules précurseurs des oligodendrocytes (OPC). Ce protocole génère Olig2 + NG2 + OPC avec un rendement élevé de 30 jours de différenciation. Nous décrivons également une méthode pour générer des &quot;dopage&quot; OPC qui peut tirer des potentiels d&#39;action.

La différenciation des cellules souches embryonnaires en précurseurs d&#39;oligodendrocytes

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

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JoVE Journal

Biology

15.2K vues.  School of Medicine, University of California, Davis. Nous décrivons une molécule à base de petites protocole pour la différenciation des cellules souches embryonnaires de souris en cellules précurseurs des oligodendrocytes (OPC). Ce protocole génère Olig2 + NG2 + OPC avec un rendement élevé de 30 jours de différenciation. Nous décrivons également une méthode pour générer des "dopage" OPC qui peut tirer des potentiels d'action.

Vidéo: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagation des souches embryonnaires humaines (ES) Cellules

Recherche

Biologie

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.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Culture de la souris de neurones précurseurs de cellules souches

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Dérivation de cellules souches hématopoïétiques de cellules souches embryonnaires murines

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprécipitation de la chromatine des cellules souches embryonnaires humaines

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Différenciation in vitro de souris embryonnaires souches (MES) Cellules utilisant la méthode de goutte suspendue

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Différenciation efficace des cellules souches embryonnaires de souris dans les neurones moteurs

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Différenciation de la peau de souris nouveau-nés des cellules souches dérivées dans les cellules germinales comme<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...