Les auteurs remercient Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, et le Stanford petit noyau d&#39;imagerie animale pour l&#39;assistance technique. Les auteurs remercient également AM Bickford, Inc pour le soutien des équipements vétérinaires. Cette recherche a été financée par des subventions de recherche du National Institutes of Health (R01 HL-75 774, R01 CA098303, R21 HL085743, 1K12 HL087746), le tabac en Californie Programme connexes Disease Research de l&#39;Université de Californie (15IT-0257 et 1514RT-0169) , et le California Institute for Regenerative Medicine (RS1-00183). NH est soutenu par une bourse de l&#39;American Heart Association. peut Heart Association.

1. La différenciation des CSE de souris en cellules endothéliales <ol><li> Le protocole de différenciation en cellules endothéliales CES est décrite ailleurs et n&#39;est pas l&#39;objet de ce protocole 2,3. Brièvement, les cellules sont autorisés à se différencier, et les cellules qui sont positifs pour les marqueurs endothéliaux tels que CD31 ou vasculaire cadhérine endothéliale (VE-cadhérine) sont ensuite purifiées par tri cellulaire activé par fluorescence (FACS). </li></ol> 2. Construction du gène rapporteur Double Fusion et transduction lentiviraux <ol><li> La bioluminescence peut être utilisé pour suivre les cellules qui sont modifiées pour exprimer des gènes rapporteurs tels que la luciférase de luciole (fluctuations). Pour cette application, nos cellules contiennent à la fois fluctuations et amélioré la protéine de fluorescence verte gène de fusion (eGFP) sous le contrôle d&#39;un promoteur interne ubiquitine. En vue de modifier les cellules, le vecteur lentiviral contenant transgène clés optimisés éléments génétiques de remplir les critères de bio-sécurité ainsi que l&#39;efficacité de transduction accrue, UFP-FG vecteur développé dans notre laboratoire portant le gène de fusion fluc-eGFP journaliste peut être utilisé de façon stable transduire les cellules après différenciation. Cette construction de fusion permet à la fois la bioluminescence et de suivi de fluorescence des cellules transplantées. La procédure pour générer des particules virales, la transduction et la production de cellules marquées que les gènes rapporteurs express est décrit ailleurs 4. </li></ol> 3. La transplantation de l&#39;ESC-dérivées des cellules endothéliales de la patte postérieure ischémique <ol><li> Commencez la procédure en préparant une souris qui a subi une ischémie des membres postérieurs pour la transplantation de cellules. Pour ce faire, placez la souris dans la chambre de l&#39;induction anesthésique contenant 1-3% d&#39;isoflurane dans 100% d&#39;oxygène à un débit d&#39;1l/min. </li><li> Laisser la souris dans la chambre de l&#39;induction jusqu&#39;à ce qu&#39;il soit insensible aux stimuli extérieurs. Puis enlever l&#39;animal de la chambre de l&#39;induction. </li><li> Ensuite, placer l&#39;animal dans la position couchée sur la table d&#39;opération et de le connecter à un flux continu de 1-3% d&#39;isoflurane dans 100% d&#39;oxygène à un débit d&#39;1l/min. </li><li> Essuyer la peau de la patte postérieure avec trois gommages bétadine et de l&#39;alcool en alternance. </li><li> Une fois que la peau est nettoyée, obtenir un million ESC dérivées des cellules endothéliales dans 30 ul de tampon phosphate salin (PBS). Chargez ces cellules dans une aiguille de calibre 28. </li><li> Lorsque les cellules sont prêtes, soulevez doucement et d&#39;étendre la patte arrière pour mieux visualiser l&#39;emplacement du muscle gastrocnémien. Alors que la jambe est prolongée, insérez l&#39;aiguille à travers la peau dans le muscle sous-jacent. Prenez soin de ne pas s&#39;approcher de l&#39;os. Doucement et injecter lentement le mélange de cellules 30 uL dans le gastrocnémien. Pour une injection intramusculaire, 30 uL est proche de la limite de volume qui peut être injecté. Par conséquent, de calibre 28 seringues à insuline sont préférés car, dans notre expérience, ils éliminent le volume de perte dans les aiguilles de seringues. </li><li> Après l&#39;injection est terminée, retournez la souris dans la cage de récupération et de le surveiller en continu jusqu&#39;à éveillé. Permettre à l&#39;animal de récupérer pendant plusieurs heures et ensuite procéder à l&#39;en imagerie par bioluminescence in vivo des cellules transplantées. </li></ol> 4. L&#39;imagerie par bioluminescence de l&#39;ESC-dérivée des cellules endothéliales in vivo <ol><li> L&#39;ESC transplanté des cellules dérivées endothéliales ont été modifiées pour exprimer les fluctuations et le gène de fusion EGFP sous le contrôle d&#39;un promoteur interne ubiquitine. Par conséquent, la bioluminescence peut être utilisé pour suivre les cellules au sein du membre postérieur ischémique. </li><li> Pour commencer cette étape, mettez le système d&#39;imagerie par bioluminescence et du logiciel d&#39;acquisition image vivante. Puis initialiser le système d&#39;acquisition et de spécifier les dimensions du champ de vision. </li><li> Ensuite, placez le papier noir mat sur la boîte à absorber les émissions d&#39;imagerie de fond. </li><li> Une fois la boîte d&#39;imagerie est prêt, placer la souris dans la chambre de l&#39;induction anesthésique contenant 1-3% d&#39;isoflurane dans de l&#39;oxygène à la sortie de 1l/min. Laisser la souris dans la chambre de l&#39;induction jusqu&#39;à ce qu&#39;il soit insensible aux stimuli extérieurs. Puis enlever l&#39;animal de la chambre de l&#39;induction. </li><li> Enlever les poils des deux membres postérieurs en utilisant un rasoir électrique, si nécessaire. </li><li> Injecter 10 uL de D-luciférine par gramme de poids corporel dans le péritoine. Le D-luciférine est préparé à l&#39;avance dans les solutions de stock filtrée de 15 mg / ml dans le PBS. </li><li> Une fois la luciférine est injecté, placer l&#39;animal dans la boîte d&#39;imagerie sur le papier noir en position couchée, connecté à un flux continu de l&#39;isoflurane. </li><li> Placer les animaux dans la boîte d&#39;imagerie et de la connexion à l&#39;isoflurane. </li><li> Commencez à acquérir des images pendant 10-60 secondes, afin de déterminer un temps d&#39;exposition optimal pour lequel l&#39;image n&#39;est pas saturé. Si l&#39;image devient saturé, de réduire le temps d&#39;exposition. Si le signal de bioluminescence est très faible, augmenter le temps d&#39;exposition. </li><li> Using le temps d&#39;exposition optimale, poursuivre l&#39;acquisition de photos à chaque 1-3 minutes jusqu&#39;à ce que le signal atteint le maximum, puis se fane. Lorsque l&#39;acquisition est terminée, enregistrez le fichier. </li><li> Pour analyser les données, sélectionner des régions d&#39;intérêt (ROI) qui couvrent le site d&#39;injection. Comme témoin négatif, un retour sur investissement similaires peuvent être sélectionnées pour la jambe non opérée. En utilisant le logiciel, mesurer la radiance totale, qui est exprimée en unités de photons / seconde / cm 2 / stéradian (P / S / cm 2 / sr), pour le ROI pour chaque point temporel. La valeur maximale doit être utilisé dans les données finales. Les données peuvent également être exportées vers Excel pour une utilisation future. </li><li> Une fois toutes les données sont acquises, le retour à l&#39;animal de la cage de récupération et de surveiller en permanence jusqu&#39;à ce que le réveil des animaux. </li><li> Répétez cette procédure pour suivre les cellules au cours du temps. </li><li> A un point de temps désiré, l&#39;animal peut être euthanasiés pour l&#39;évaluation de la fonction des tissus. </li></ol> 5. Les résultats représentatifs <img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="Figure 1" width="600" height="600" /> Une image représentant la bioluminescence des cellules transplantées dans le membre postérieur gauche ischémique est montré dans la figure 1. Pendant l&#39;acquisition de la bioluminescence, l&#39;intensité va augmenter avec le temps, et la valeur maximale obtenue au cours du temps doivent être déclarés comme la valeur finale.

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

CES sont une source de cellules prometteuses pour le traitement de l&#39;ischémie tissulaire en raison de leur plasticité de différenciation et de leur capacité à donner naissance à des lignées de cellules comprenant les trois feuillets embryonnaires, dont les cellules endothéliales. Pour surmonter les problèmes éthiques associés avec les CES, cellules souches pluripotentes induites (CISP) peut être une source de cellules souches pluripotentes alternatives qui surmonte les préoccupations éthiques. Outre les CES, les cellules s...

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		<th>Comment</th>
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<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
<td>&nbsp;</td>
<td>28G insulin syringe is preferred</td>
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<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
<td>&nbsp;</td>
<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
</tr>
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<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
<td>Equipment</td>
<td>Xenogen Corporation</td>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

Artérielle périphérique maladie (PAD) les résultats de rétrécissement des artères périphériques qui fournissent le sang oxygéné et de nutriments pour les jambes et les pieds, cette pathologie entraîne des symptômes tels que la claudication intermittente (douleur à la marche), des ulcérations douloureuses ischémiques, ou même menaçant le membre gangrène. On croit généralement que l&#39;endothélium vasculaire, une monocouche de cellules endothéliales qui investit la surface luminale de tous les vaisseaux sanguins et lymphatiques, joue un rôle dominant dans l&#39;homéostasie vasculaire et la régénération vasculaire. En conséquence, base de cellules souches de régénération de l&#39;endothélium peut être une approche prometteuse pour le traitement de la MAP. Dans cette vidéo, nous démontrons la transplantation de cellules souches embryonnaires (CSE) dérivées des cellules endothéliales pour le traitement de l&#39;ischémie unilatérale hindimb comme un modèle de la PAD, suivi par des non-invasive de la cellule de suivi homing et de la survie par imagerie par bioluminescence. Les matériaux et les procédures spécifiques pour la livraison et l&#39;imagerie cellulaire seront décrites. Ce protocole suit une autre publication dans la description de l&#39;induction d&#39;une ischémie des membres postérieurs par Niiyama et al. 1

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

La procédure chirurgicale pour la livraison de cellules souches embryonnaires dérivées des cellules endothéliales de la patte postérieure ischémique est démontrée, avec des non-invasive de suivi par imagerie par bioluminescence.

Les cellules souches embryonnaires des cellules dérivées endothéliales pour traitement de l&#39;ischémie des membres postérieurs

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K vues.  Stanford University. La procédure chirurgicale pour la livraison de cellules souches embryonnaires dérivées des cellules endothéliales de la patte postérieure ischémique est démontrée, avec des non-invasive de suivi par imagerie par bioluminescence.

Vidéo: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagation des souches embryonnaires humaines (ES) Cellules

Recherche

Biologie

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Palpage pour activité complexe mitochondrial dans les cellules souches embryonnaires humaines

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Modèle murin d&#39;ischémie des membres postérieurs

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Préparation Étaler chromosomique des cellules souches embryonnaires humaines pour le caryotype

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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 (&gt;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 NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

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 ...

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 ...