Ce travail a été soutenu par la Fondation Alexander von Humboldt (5090371), le Centre Kühne Christine de recherche sur les allergies et l&#39;éducation (CK-CARE), et le Bayerischen Staatsministerium (pour KD).

1. Isolation SKP partir des cultures de fibroblastes primaires <ol><li> des cultures de fibroblastes ou l&#39;autre des banques de cellules ou directement obtenu à partir de biopsies de peau sont maintenues en culture en croissance des fibroblastes du milieu DMEM contenant 15% de sérum de veau foetal, 2 mM de glutamine, 10 mg / ml de pénicilline et 10 mg / ml de streptomycine. </li></ol> Fibroblastes humains GMO3349C et GMO8398A ont été obtenus à partir de l&#39;Institut Coriell pour la recherche médicale (Camden, New Jersey) et ont été utilisés dans cette étude. <ol start="2"><li> Cultures de doublements de population (PPD) 20 à 35 ont été utilisés pour les cultures SKP à une confluence de 80%. Une boîte de 10 cm de culture tissulaire (BD Falcon) contient environ 1,5.10 6 cellules. </li><li> Laver les cellules avec du PBS et incuber avec 2 ml de solution de trypsine (0,25%, Invitrogen) pendant 1 heure à 37 ° C. </li><li> Recueillir les cellules à partir de la plaque avec 5 ml de PBS et transférer la suspension de cellules dans un tube Falcon de 15 ml. </li><li> Incuber les cellules à 4 ° C pendant 24 heures. </li><li> Préparer le milieu de croissance SKP composé de DMEM-F12, 3:1 (V / V) et 40 ng / ml FGF2 (BD Biosciences, 20 ng / ml d&#39;EGF (BD Biosciences), B27 sérique supplément gratuit de 2% (Invitrogen), 1 ug / ml Fungizone (Invitrogen) et 25 um / ml gentamicine (Invitrogen). </li><li> Pellet les cellules à 1200 rpm pendant 5 min et remettre le culot cellulaire directement dans le milieu de croissance SKP 4 ml. Transférer la suspension cellulaire à un cm2 flacon de culture de tissus 25 (BD Falcon). </li><li> Incuber le ballon à 37 ° C et à surveiller la culture pour la formation de la sphère par jour pendant 3 à 4 semaines. </li></ol> 2. Sphère Conditions de culture <ol><li> les cellules de la culture dans un flacon de 25 cm2 (BD Falcon) à 37 ° C, 5% de CO 2. </li><li> Agiter le flacon vigoureusement tous les jours pour éviter les cellules d&#39;adhérer au fond du flacon. Si nécessaire, une pipette de haut en bas avec un 2 ml pipette stérile pour détacher les cellules adhérentes et transférer la culture dans un nouveau flacon. </li><li> Premières sphères commencent à construire dans les 3 to 4 jours. </li><li> Soit sphères sédiment au fond du flacon et le changement de la moitié du milieu tous les 3 jours. Pour conserver la même concentration finale de facteurs de croissance les facteurs de croissance ajouter 2X au milieu de croissance de SKP fraîchement préparée. </li><li> Maintenez le volume constant maximum de 4 ml. </li><li> Quand sphères atteignent une taille d&#39;environ 200 mm, ils doivent être repiquées en brisant les sphères de petite taille par vigoureuse pipetage de haut en bas avec une pipette de 2 ml. </li><li> La culture est divisé en deux flacons de 25 cm2 (BD Falcon) et des sphères cultures sont alimentation comme décrit dans l&#39;étape 2.4). </li><li> Sphères sont collectées pour des marqueurs de cellules souches dépistage par jour 16 à 21. </li></ol> La procédure de 2.6) et 2.7) décrivent la propagation des sphères à (1) permet de nutriments et de facteurs de croissance dans du milieu compris SKP d&#39;accéder à toutes les cellules au sein de chaque sphère et (2) pour développer la culture de la sphère SKP avant utilisation. Typiquement cultures Sphère sous nos cuconditions de LTURE maintenir la capacité de croissance sur une période de trois mois et sont repiquées entre 3 à 4 fois. 3. Immunocytochemistry de cellules souches Marqueurs <ol><li> cultures de Sphère sont récoltées dans du PBS par jour 16 à 21. Les cultures sont culottés à 1000 rpm pendant 5 min. </li><li> Sphères sont remises en suspension dans un petit volume de PBS. </li><li> Dessinez deux cercles avec un stylo DAKO d&#39;environ 0,5 m de diamètre sur des lames de microscope (marque Fisher). </li><li> Ajouter 50 ul de suspension de la sphère dans les cercles. </li><li> Voir par microscopie optique la présence de sphères dans chaque goutte. </li><li> Que les gouttes sec sous le capot. </li><li> Soit geler les diapositives à -80 ° C ou procéder avec le protocole de coloration par immunofluorescence. </li><li> Par immunofluorescence, fixer les lames avec du méthanol pré-refroidi (100%) à -20 ° C pendant 10 min. </li><li> Laver les lames avec PBS. </li><li> Bloquer les diapositives avec PBS/10% de sérum de veau fœtal / 0,02% de Triton X100pendant au moins 1 heure. </li><li> Incuber avec l&#39;anticorps primaire (par exemple, anti-Nestin, anti-Oct4, anti-TG30, les anticorps anti-Nanog, tableau 3) dilué dans du tampon de blocage: PBS/10% de FBS pendant 2 heures à température ambiante ou une nuit à 4 ° C. </li><li> Laver 3 fois avec le tampon de blocage et incuber avec l&#39;anticorps secondaire pendant 1 h à température ambiante. </li><li> Laver 3 fois avec le tampon de blocage et 3 fois avec PBS. </li><li> Montage diapositives avec montage moyen Vectashield (Vector Inc.). </li><li> Analyser des échantillons pour l&#39;expression de marqueurs de cellules souches par immunofluorescence. </li></ol> 4. Analyse PCR en temps réel des niveaux d&#39;expression de marqueurs de cellules souches <ol><li> Prenez environ 2-3 cultures Sphère ml de jour de 18 à 21. </li><li> Pellet les sphères à 1200 rpm pendant 5 min et aspirer tout moyen. </li><li> Isoler l&#39;ARN à partir de sphères en utilisant le Minikit RNeasy (Qiagen, Valencia, CA). </li><li> Évaluer l&#39;ARN pureté par spectrométrie et gel d&#39;agarose. &lt;/ Li&gt; <li> Synthétiser l&#39;ADNc (Omniscript de la transcriptase inverse (Qiagen)) en utilisant l&#39;ARN cellulaire total comme matrice. </li><li> Valider les marqueurs de cellules souches par Realtime-PCR utilisant des amorces pour les marqueurs des cellules souches (voir tableau 1) en une puissance SYBR Green Mastermix PCR (Applied Biosystems) avec une concentration de 375 nM de chaque amorce et 50 ng de matrice dans un 20 pi volume de réaction. GAPDH est utilisé comme contrôle endogène. </li><li> Pour l&#39;amplification utilise une dénaturation initiale à 95 ° C pendant 2,5 min suivie de 40 cycles à 95 ° C pendant 5 s et 60 ° C pendant 20 s. </li><li> Analyser la course avec les 2 (ΔΔCt) méthode 8. </li></ol> 5. Différenciation Réalisé dans les cellules musculaires lisses <ol><li> Sphères du jour 21 à 26 ont été étalées en 6 boîtes de culture puits (BD Falcon) en milieu SKP. </li><li> Les cellules ont été autorisés à adhérer et à dépasser des sphères en milieu SKP pendant 72 heures. </li><li> Les cellules musculaires lisses (CML) differentiation commencé initiée par remplacé le milieu par un milieu de différenciation SMC consistant en haut glucose milieu de Eagle modifié par Dulbecco (Invitrogen) contenant 5% de SVF, de 5 ng / ml de PDGF-BB (Invitrogen), et de 2,5 ng / ml de TGF-b1 (Invitrogen) . </li><li> Le milieu a été changé tous les 3 jours avec un milieu de différenciation SMC fraîchement préparé pour une période de 3 à 4 semaines dans les cultures. </li><li> Dépistage des marqueurs SMC a été réalisée après 3 à 4 semaines par immunohistochimie. </li><li> Les cellules ont été fixées avec une solution de paraformaldéhyde 4% dans du PBS pendant 10 min. </li><li> Les cellules ont été perméabilisées avec du PBS contenant 0,3% de Triton X-100 pendant 30 min. </li><li> Les cellules ont été incubées avec des anticorps contre αSMA (MO851, Dako, 1:100), ou calponine (M3556, Dako, 1:100) pendant 1 heure à température ambiante et traité comme décrit dans 3.12) à 3.15). </li></ol>

<ol>
	<li>Jahoda, C. A., Whitehouse, J., Reynolds, A. J., Hole, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+dermal+cells+differentiate+into+adipogenic+and+osteogenic+lineages.">Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages.</a> Exp. Dermatol. 12, 849 (2003).</li><li>Watt, F. M., Celso, L. o., C, V., Silva-Vargas, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells:+an+update.">Epidermal stem cells: an update.</a> Curr. Opin. Genet. Dev. 16, 518 (2006).</li><li>Blanpain, C., Horsley, V., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells:+turning+over+new+leaves.">Epithelial stem cells: turning over new leaves.</a> Cell. 128, 445 (2007).</li><li>Hunt, D. P., Jahoda, C., Chandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+skin-derived+precursors:+from+biology+to+clinical+translation.">Multipotent skin-derived precursors: from biology to clinical translation.</a> Vurr. Opin. Biotechnol. 20, 522 (2009).</li><li>Toma, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat. Cell Biol. 3, 778 (2001).</li><li>Fernandes, K. J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dermal+niche+for+multipotent+adult+skin-derived+precursor+cells.">A dermal niche for multipotent adult skin-derived precursor cells.</a> Nat. Cell Biol. 6, 1082 (2004).</li><li>Wenzel, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+adult+stem+cells+from+patients+with+Hutchinson-Gilford+progeria+syndrome+express+low+levels+of+progerin+in+vivo.">Na&#239;ve adult stem cells from patients with Hutchinson-Gilford progeria syndrome express low levels of progerin in vivo.</a> Bio. Open. 1, (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402 (2001).</li><li>Biernaskie, J. A., McKenzie, I. A., Toma, J. T., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skin-derived+precursors+(SKPs)+and+differentiation+and+enrichment+of+their+Schwann+cell+progeny.">Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny.</a> Nature Protocol. 1, 2803 (2006).</li><li>Toma, J. G., McKenzie, I., Bagli, D., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+skin-derived+precursors+from+human+skin.">Isolation and characterization of multipotent skin-derived precursors from human skin.</a> Stem Cells. 23, 727 (2005).</li><li>Fernandes, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+neurogenic+potential+of+multipotent+skin-derived+precursors.">Analysis of the neurogenic potential of multipotent skin-derived precursors.</a> Electrophoresis. 201, 32 (2006).</li><li>Hill, l. K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+vascular+progenitor+cells+capable+of+endothelial+and+smooth+muscle+cell+function.">Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function.</a> Exp. Hematol. 38, 246 (2010).</li></ol>

Nous n&#39;avons rien à révéler.

En utilisant la méthode décrite ici, les cellules souches dermiques naïfs peuvent être isolés à partir de cultures de fibroblastes dermiques primaires. Grâce à cette approche, nous avons récemment signalé l&#39;isolement et la caractérisation des cellules souches adultes à partir de cultures de fibroblastes provenant de patients atteints d&#39;un syndrome génétique rare, Hutchinson-Gilford progeria syndrome 7. Comme le montrent ici les cellules marqueurs de cellules souches expriment précurseur...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Invitrogen</td>
			<td>31966-047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Invitrogen</td>
			<td>21885-108</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10270-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td>Final conc.: 200 mM</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>Final conc.: 10 mg/ml /10 mg /ml</td>
		</tr>
		<tr>
			<td>trypsin solution (0.25%)</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mixture (Ham)</td>
			<td>Invitrogen</td>
			<td>21765-029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>BD Biosciences</td>
			<td>4114-TC-01M</td>
			<td>Final conc.: 40 ng/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>236-EG-200</td>
			<td>Final conc.: 20 ng/ml</td>
		</tr>
		<tr>
			<td>PDGFBB</td>
			<td>Invitrogen</td>
			<td>PHG0043</td>
			<td>Final conc.: 5 ng/ml</td>
		</tr>
		<tr>
			<td>TGF-b1</td>
			<td>Invitrogen</td>
			<td>PHG9204</td>
			<td>Final conc.: 2.5 ng/ml</td>
		</tr>
		<tr>
			<td>25 cm2 flask</td>
			<td>Omnilab</td>
			<td>FALC353109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS w/o CaMg</td>
			<td>Invitrogen</td>
			<td>14190-169</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Roth</td>
			<td>8388.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Inc.</td>
			<td>H-1200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy Minikit</td>
			<td>Qiagen, Valencia, CA</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Omniscript Reverse Transcriptase</td>
			<td>Qiagen</td>
			<td>205113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SsoFast EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>172-5201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td>Final conc.:1 mg/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Specific reagents and equipment.</td>
		</tr>
	</tbody>
</table>

na&iuml;ve adult stem cells isolation from primary human fibroblast cultures

Au cours de la dernière décennie, plusieurs populations de cellules souches adultes ont été identifiés dans la peau humaine 1-4. L&#39;isolement des précurseurs dermiques adultes multipotentes a d&#39;abord été rapporté par Miller F. D laboratoire 5, 6. Ces premières études ont décrit une population de cellules précurseurs pluripotentes à partir de derme des mammifères adultes 5. Ces cellules - SKP appelés, pour les précurseurs dérivés de la peau - ont été isolés et sont passées de rongeurs et de la peau humaine et différenciées en neurones et deux descendants du mésoderme, y compris les types de cellules jamais trouvés dans la peau, comme les neurones 5. Des études immunocytochimiques sur SKPs culture ont révélé que les cellules expriment la vimentine et la nestine, une protéine de filament intermédiaire exprimé des précurseurs neuronaux et musculaires squelettiques, en plus de la fibronectine et des marqueurs de cellules souches multipotentes 6. Jusqu&#39;à présent, les cellules souches SKPs la population adulte ont été isolés à partir de biopsies de peau de mammifère fraîchement récoltées. ve_content &quot;&gt; Récemment, nous avons mis en place et a indiqué que la population de la peau des cellules précurseurs dérivées pourrait rester présente dans des cultures de fibroblastes primaires établies à partir de biopsies de la peau 7. L&#39;hypothèse selon laquelle un petit nombre de cellules souches somatiques pourraient résider dans des cultures de fibroblastes primaires de doublements de population au début était basées sur les observations suivantes: (1) SKPs et les cultures de fibroblastes primaires proviennent du derme, et donc un petit nombre de cellules SKP pourraient rester présente dans les cultures de fibroblastes dermiques primaires et (2) des cultures de fibroblastes primaires cultivées à partir de portions congelées qui ont été soumis à une température défavorable pendant le stockage ou le transfert contenait un petit nombre de cellules qui sont restées viables 7. Ces cellules rares étaient capables de développer et pourrait être repiquées plusieurs fois. Cette observation suggère qu&#39;un petit nombre de cellules avec une puissance de prolifération élevé et la résistance au stress étaient présents dans les cultures de fibroblastes humains 7. Nous avons profité de ces résultats pour établir un protocole pour l&#39;isolement rapide des cellules souches adultes à partir de cultures de fibroblastes primaires qui sont facilement disponibles à partir de banques de tissus à travers le monde (Figure 1). Cette méthode a une signification importante car elle permet d&#39;isoler des cellules précurseurs lorsque des échantillons de peau ne sont pas accessibles alors que les cultures de fibroblastes peuvent être disponibles à partir de banques de tissus, ainsi, ouvrir de nouvelles opportunités pour disséquer les mécanismes moléculaires qui sous-tendent les maladies génétiques rares ainsi que les maladies de modélisation dans un Antenne.

Nous montrons qu&#39;une population de cellules qui augmentent de manière sélective pour générer des sphères SKP sous condition d&#39;une croissance maîtrisée, comprenant des EGF et FGF2 sont présents dans les cultures de fibroblastes dermiques primaires (Figure 1) Comme nous l&#39;indiquions récemment 7. des cultures de fibroblastes de PPD 15 à 25, qui correspondent généralement aux souches de fibroblastes primaires disponibles à partir de banques de...

Watch this Scientific Journal Video about NaÃ¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures at JoVE.com

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

Nous rapportons une méthode pour isoler les cellules naïves multipotentes précurseurs dérivés de la peau (SKP) à partir de cultures de fibroblastes humains primaires. Nous montrons que ces SKPs issus de cultures de fibroblastes partagent des propriétés semblables des cellules souches à celles provenant directement de biopsies de peau humaine. Ces cellules expriment le marqueur de la crête neurale, nestine, en plus des marqueurs tels que OCT4 multipotentes et 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 ...

nave-adult-stem-cells-isolation-from-primary-human-fibroblast-cultures

Research

JoVE Journal

Biology

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

14.0K vues.  Technische Universität München. Nous rapportons une méthode pour isoler les cellules naïves multipotentes précurseurs dérivés de la peau (SKP) à partir de cultures de fibroblastes humains primaires. Nous montrons que ces SKPs issus de cultures de fibroblastes partagent des propriétés semblables des cellules souches à celles provenant directement de biopsies de peau humaine. Ces cellules expriment le marqueur de la crête neurale, nestine, en plus des marqueurs tels que OCT4 multipotentes et Nan...

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

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

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 ...

Recherche

Biologie

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Établir des cultures primaires de fibroblastes adultes à partir de rongeurs

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Préparation des cellules de souris embryonnaires fibroblastiques conviennent pour la culture embryonnaires humaines et cellules souches pluripotentes induites

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Suppression des fibroblastes rapide de haute densité embryonnaires humaines cultures de cellules souches

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolement des cellules immunitaires des tumeurs primitives

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolation Manuel des cellules souches du tissu adipeux de Lipoaspirates humaines

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...