Les auteurs remercient le Dr Katharina Schallmoser pour fournir pHPL et Margaretha Frühwirth et Daniela Thaler pour une assistance technique excellente.

A.) ECFC ISOLATON JOUR 1: <ol><li> Avant de commencer avec l&#39;expérience de préparer la croissance milieu de culture cellulaire endothéliale moyenne-2 (EGM-2). Supplément 500ml endothéliale Basal Medium-2 avec de l&#39;héparine (1ml de 1000 U / ml), solution de 5ml 100x de pénicilline / streptomycine (tous Sigma, St. Louis, MO; <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamine (2 mM de concentration finale ), et 50 ml commun lysat plaquettaire humain (pHPL). Ensuite, ajoutez 0,2 hydrocortisone ml, 2 ml hFGF-B, 0,5 ml de VEGF, EGF 0,5 ml, 0,5 ml d&#39;IGF-1 et 0,5 ml d&#39;acide ascorbique (à condition que SingleQuots, tous Lonza, Walkersville, MD; <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience. com /</a> ). Filtrat stérile de la moyenne grâce à un 0,2 m-taille des pores du filtre 500 ml sous vide Millipore. Depuis pHPL formes petits caillots vous pouvez avoir besoin de deux filtres pour une moyenne. Préchauffer filtrée EGM-2 à 37 ° C dans un bain d&#39;eau </li><li> Recueillir des échantillons de sang par le dessin 5 ml de sang périphérique adulte humain de la veine cubitale dans un sans conservateur 6ml de sodium d&#39;héparine tube Vacutainer sang. Vous devez traiter les échantillons de sang dans un délai maximum de deux heures. </li><li> Commencez par préparer un 75 cm 2 (T75; Costar) fiole avec un bouchon à évent, une pipette 25 ml et deux pipettes 5 ml dans un banc de flux laminaire. </li><li> Pré-remplissage de 15 ml pré-chauffé complété EGM-2 dans le ballon avec la pipette de 25 ml. Maintenant, ouvrez le flacon d&#39;échantillon de sang et tous les 5 ml à la pipette 5 ml dans le flacon. Rincer la fiole de sang vides avec 5 ml d&#39;supplémentaires EGM-2 avec la seconde pipette 5 ml. Le volume total au sein du T75 est de 25 ml. Fermez le bouchon à évent-du flacon et le placer dans une étuve à 37 ° C, 5% de CO 2 et 95% d&#39;humidité pendant 24 heures. </li></ol> JOUR 2: <ol><li> Après environ 24 heures (une nuit), vous devriez retirer le mélange du sang moyen de se débarrasser des cellules non adhérentes. Pour préparer ces deux pipettes 25 ml et trois 5 pipettes ml dans le flux laminaire, préchauffer de 20 ml de compléter EGM-2 (préparée le jour précédent) et 30 ml de PBS à 37 ° C dans un bain d&#39;eau. </li><li> Retirer le ballon avec l&#39;échantillon de l&#39;incubateur et supprimer tous les surnageant avec une pipette de 25 ml. Prenez soin de ne pas rayer la surface flacon pour éviter d&#39;endommager toute colonie potentiels. Lavez la surface intérieure des cellules flacon adhésion à trois reprises par délicatement en ajoutant 10 ml de PBS préchauffé et en inclinant le flacon d&#39;un côté à l&#39;autre. Retirez le CPE immédiatement à chaque fois et les jeter. </li><li> Ajouter 20 ml de frais préchauffé EGM-2 dans le ballon et le replacer dans l&#39;incubateur. Ce processus devrait être effectué aussi rapidement que possible et en douceur pour les cellules attachée à ne pas se détacher. CPEF détachent facilement si elles sont conservées dans du PBS ou à température ambiante pendant trop longtemps. </li><li> Ecran à travers le ballon systémique tous les deux jours à partir de deux jours en utilisant un faible grossissement d&#39;un microscope optique à la recherche d&#39;excroissance ECFC. Quand une colonie se trouve la marque sur le dessus sur le côté extérieur de la fiole en indiquant la position de la colonie. Notez que le Flaks ne doit pas être en dehors de l&#39;incubateur pendant plus de 5 minutes. </li></ol> JOUR 5: <ol><li> Le cinquième jour après le semis effectuer un changement de milieu complet pour enlever des cellules non adhérentes. Pour ce faire préchauffer 20 ml d&#39;EGM-2 à 37 ° C dans un bain d&#39;eau et de préparer deux pipettes 25 ml. </li><li> Retirez le support complet à l&#39;aide d&#39;une pipette de 25 ml et ajouter frais, préchauffé EGM-2 complété dès que possible. Les cellules ne devraient pas être laissés sans moyen de plus d&#39;une minute pour éviter le dessèchement. Placez de nouveau dans l&#39;incubateur et répéter le test du ballon quotidien. </li><li> Alimenter les cellules en remplaçant 30% de la moyenne par les frais complété EGM-2 deux fois par semaine. </li></ol> JOUR 14-28: <ol><li> Les colonies typiques de morphologie pavées devrait apparaître autour de 12 jours. Marquer le côté supérieur extérieur de la fiole d&#39;indiquer la position de chaque colonie. Autoriser les colonies de croître jusqu&#39;à ce jour 28 cellules individuelles à moins de commencer à se détacher. Une fois, des colonies primaires ont été identifiés, ils peuvent être récoltés entre 14 et 28 jours, en fonction de leur taille. </li></ol> B.) ECFC MOISSON <ol><li> Pré-chaud 5 ml de trypsine 0,05% / EDTA, 20 ml de PBS et 5ml frais complété EGM-2. </li><li> Complètement éliminer le milieu de la fiole dans un tube Falcon (prendre soin de ne pas toucher la surface du flacon pour éviter d&#39;endommager toute colonie potentiel) et se laver soigneusement les cellules avec 10 ml de PBS préchauffé. </li><li> Ajouter 5 ml de trypsine / EDTA et incuber pendant 5 minutes dans l&#39;incubateur. Les cellules ne devraient pas être exposés à la trypsine pendant plus de 7 min, car il devient toxique. Bien tapotant les côtés de la fiole aide les cellules à se détacher. Quench activité de la protéase trypsine en ajoutant environ 10 ml du milieu de culture utilisé préservée. </li><li> Retirez la suspension cellulaire dans un Falco 50 mlTube n. Laver le ballon une fois avec 10 ml de PBS qui est ensuite ajouté à la suspension de cellules récoltées. Centrifuger la suspension cellulaire à 300 g pendant 7 min à 4 ° C pour collecter les cellules et enlever la trypsine. Jeter le surnageant et remettre le culot cellulaire immédiatement dans 1 ml de PBS. Restez sur la glace. </li><li> Déterminer le nombre de cellules comme décrit dans un protocole JoVE par R. Ricardo et K. Phelani 5. </li></ol> C) ECFC EXPANSION <ol><li> Supplément de 500 ml EGM-2 tel que décrit dans A 1.1). Préchauffer à 37 ° C dans un bain d&#39;eau. </li><li> Pour environ 2500 cm 2 d&#39;espace de culture de préparer une usine soit 11 T220 (225 cm 2) ou 15 T175 (175 cm 2) ou une cellule de quatre couches (FC-4; Nunc, Chicago, IL; <a href="http://www.nuncbrand.com/">http://www.nuncbrand . com</a> ). En outre, vous aurez besoin de deux nouvelles évent-bouchons et un entonnoir stérile spécial dans le banc d&#39;écoulement laminaire. </li><li> Pour CPEF semences à une densité cellulaire initiale de 100 c / cm 2 de calcul du nombre de cellules est nécessaire. Pour un CF-4 252 800 cellules sont resuspendues dans 500 ml fraîche EGM-2 et versé dans les FC-4 en utilisant l&#39;entonnoir stérile. Fermer un évent à l&#39;évent de plafonnement et d&#39;incliner le CF-4 d&#39;un côté de longitude, ce qui permet une répartition égale du milieu entre les 4 farines. Puis inclinez la chambre arrière et ouvrez le couvercle de l&#39;évent-bouchon. Placez CF-4 dans l&#39;incubateur. </li><li> Changer 20% de la moyenne au moins une fois par semaine jusqu&#39;à FC-4 est confluente. </li><li> Récolte des cellules comme décrit dans B.) en utilisant 70 ml de trypsine / EDTA et environ 200 ml de PBS. </li></ol> Les résultats représentatifs En utilisant cette méthode d&#39;isolement, nous avons observé une formation de colonies primaires au jour 12. Dans une étude précédente ont été en mesure de récupérer une moyenne de 4,0 ± 0,8 ECFC-colonies issues de chaque ml de sang périphérique. 4 CPEF a montré un aspect typique en pierre de galets. Après l&#39;expansion à grande échelle, avec une densité de semis de 100 cellules / cm 2, nous avons obtenu&gt; 1 x 10 8 cellules pour un total d&#39;environ 30 jours à partir de 3 hommes et 1 femmes volontaires (âge compris entre 26 et 50 ans). ECFC cultivés dans ces conditions se sont avérés être proliférative, fonctionnelle et génomiquement stable. 4 En outre les cellules peuvent être stockés (dans 10% de DMSO) dans l&#39;azote liquide pour une utilisation ultérieure. 3,4

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

Cet isolement roman et la méthode d&#39;extension est un processus simple et efficace qui est plus efficace et moins de temps en évitant toute séparation des cellules (pas de gradient de densité nécessaire) et moins coûteux que les techniques conventionnelles. En utilisant de contact pHPL aux antigènes putatifs bovine et xenoimmunization ultérieure est exclue. Pendant la période de la culture des petits caillots peuvent se former, causée par la pHPL. Basé sur notre expérience de ces caillots n&#39;affectent ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Endothelial basal Medium-2</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>500ml</td>
</tr>
<tr>
<td>EGM-2 SingleQuots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td>
</tr>
<tr>
<td>preservative-free Heparin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>&nbsp;</td>
<td>10U/ml per EGM-2</td>
</tr>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>2mM per EGM-2</td>
</tr>
<tr>
<td>Penicillin and Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>100U/mL Penicillin and 100&mu;g/mL Streptomycin per EGM-2</td>
</tr>
<tr>
<td>Trypsin/1mM EDTA </td>
<td>&nbsp;</td>
<td>Gibco Cell Culture, Invitrogen </td>
<td>&nbsp;</td>
<td>7ml for T75 flask 70ml for CF-4</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Four-layered cell factory (CF-4) </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Funnel</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T75cm2 culture flask (with vented cap)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 mL vacutainer tubes (preservative-free sodium heparin) </td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>preloaded with 108 IU/6ml of preservative-free sodium heparin</td>
</tr>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Squeezer</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0,2 pore size sterile filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>2x 500ml per EGM-2</td>
</tr>		</tbody>
		</table>
		</div>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

Ce document présente une stratégie de rétablissement pour le roman de cellules progénitrices endothéliales formant colonie (CPEF) de l&#39;héparine mais autrement non manipulés pour adultes sang périphérique humain au sein d&#39;une moyenne de 12 jours. Après l&#39;expansion à grande échelle&gt; 1x10 8 CPEF peuvent être obtenus pour d&#39;autres essais. Avantageusement en utilisant pHPL le contact des cellules humaines avec bovin antigènes sériques peut être exclue. Par cytométrie en flux et par immunohistochimie des cellules isolées peuvent être caractérisés comme ECFC et leur fonctionnalité dans vitro pour former des structures vasculaires peuvent être testés dans un essai de Matrigel. De plus, ces cellules peuvent être injectées par voie sous cutanée dans un modèle de souris pour la forme fonctionnelle, des vaisseaux perfusés in vivo. Après l&#39;expansion à long terme et la prolifération cryoconservation, la fonction et la stabilité génomique semblent être préservées 3,4. Cet isolement des animaux en protéines libres et méthode d&#39;expansion est facilement applicable à générer une grande quantité de CPEF.

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Cellules endothéliales progénitrices formant colonie (CPEF) sont un outil prometteur pour étudier l&#39;homéostasie vasculaire et de réparation.<sup&gt; 1,2</sup&gt; Cet article présente une nouvelle méthode de sérum animal-libres pour l&#39;isolement et l&#39;expansion de ECFC partir hépariné sang adultes périphérique humain avec des mises en commun lysat plaquettaire humain (pHPL) diminue le risque de la vaccination anti-bovin.

L&#39;isolement et expansion à grande échelle des cellules adultes humaines Colony Forming progénitrices endothéliales

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

isolation-large-scale-expansion-adult-human-endothelial-colony

Research

JoVE Journal

Biology

10.6K vues.  Medical University of Graz, Austria. Cellules endothéliales progénitrices formant colonie (CPEF) sont un outil prometteur pour étudier l'homéostasie vasculaire et de réparation. 1,2 Cet article présente une nouvelle méthode de sérum animal-libres pour l'isolement et l'expansion de ECFC partir hépariné sang adultes périphérique humain avec des mises en commun lysat plaquettaire humain (pHPL) diminue le risque de la vaccination anti-bovin.

Vidéo: Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolement de cellules humaines endotheliales de veine ombilicale (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Recherche

Biologie

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Grands écrans des banques métagénomiques

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolement et animale expansion sans sérum du cordon ombilical humain dérivé cellules stromales mésenchymateuses (CSM) et des cellules endothéliales progénitrices Colony Forming (CPEF)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

L&#39;isolement des cellules endothéliales valvulaires

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolement et culture de cellules progénitrices embryonnaires aviaires valvulaire

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Un dosage quantitatif de l&#39;insuline exprimer formant colonie Progéniteurs

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Isolement des cellules CD133 + Foie souches pour l&#39;expansion clonale

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caractérisation phénotypique et fonctionnelle des cellules endothéliales formant colonie dérivés du sang de cordon ombilical humain

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...