Ce travail est financé par le NIH / NCI octroi R01CA-118916

1. Extraire des cellules souches épithéliales de la peau humaine <ol><li> Avant de commencer la procédure d&#39;isoler des cellules souches épithéliales on a besoin pour préparer les médias respectifs et des réactifs (voir tableau 1). </li><li> Fraîches peau du cuir chevelu humain adulte à partir de procédures lifting ou biopsie est recueilli, puis incuber dans FBS DMEM / 10% / dispase (4 mg / mL) pendant la nuit à 4 ° C. L&#39;incubation pendant 2-4 heures à 37 ° C est également efficace. Morceaux de peau doit être d&#39;une largeur maximale de 1 cm pour permettre l&#39;enzyme de pénétrer. </li><li> Transfert de la peau dans une boîte de Pétri stérilisées, arracher chaque poil de la peau en saisissant la tige du cheveu près de la surface de la peau et en tirant fermement et en douceur. Sélectionnez follicules au stade télogène basée sur leur morphologie sous un microscope à dissection, découpez la région renflement (figure 1A) follicules transférer dans un tube de 15 ml stérilisés. Follicules anagène peut aussi être utilisée si elle est coupée à la partie supérieure 1 / 3 du follicule pour obtenir follicule pileux &quot;renflement&quot; région (figure 1B). </li><li> Incuber les fragments isolés du follicule dans un mélange de 0,05% de trypsine-EDTA (Gibco) et Versène (0,53 mM EDTA 4NA, GIBCO) (01h01) pour 15-20 min à température ambiante avec agitation périodique, ajouter 4 ml de DMEM + 10 % de FBS pour arrêter la réaction, ralentit pendant 5 min à 800 rpm. Alternativement, des explants de tissu folliculaire fragment peut être mis en culture, sans trypsine digérer pour des cellules en culture à partir de follicules simples. </li><li> Rejeter le surnageant avec soin (~ 0,2 à 0,5 ml sauver pour éviter de perdre des cellules) et remettre en suspension dans 1 ml de KCM (moyenne des kératinocytes), sans EGF. </li></ol> 2. Culture primaire des cellules souches épithéliales <ol><li> Avant de cultiver les cellules souches épithéliales, la mitomycine C-traitée 3T3-J2 cellules (cellules nourricières) doit être préparé comme suit. 3T3-J2 cellules sont cultivées dans du DMEM avec 10% de FBS avant le traitement. Retirez le support de culture cellulaire et laver deux fois avec PBS, traiter cellule avec 15μg / ml de mitomycine C dans du DMEM sans sérum pendant 2 heures à 37 ° C, 5% de CO 2. Retirez la mitomycine C contenant du DMEM et laver deux fois avec du PBS, les cellules sont prêtes à utiliser. (Mitomycine C-traitée 3T3-J2 cellules peuvent également être préparés précédemment et stocké à -80 ° C. Dégel et les graines des cellules un jour avant culture primaire des cellules souches épithéliales, incuber à 37 ° C, 5% de CO 2. 150,000-200,000 3T3-J2 cellules par puits de plaque de 6 puits est recommandée. </li><li> Graine cellule isolée tige follicule pileux de l&#39;étape 1.5 sur mitomycine C-traitée 3T3-J2 cellules dans KCM, sans la nuit à 37 ° C EGF, 5% de CO 2, a changé d&#39;EGF-KCM contenant le lendemain. Les cellules sont cultivées à 37 ° C dans une atmosphère humide contenant 5% de CO 2. Tous les kératinocytes sont nourris avec KCM contenant EGF tous les 2 jours et cultivés pendant 14-20 jours. Vérifiez la cellule chaque jour sous le microscope. Cellules souches du follicule pileux va former des colonies (figure 2A). Explants follicule pileux se forme des excroissances après 10-14 jours (figure 2B). </li></ol> 3. Cellules souches épithéliales Passage <ol><li> Laver les cellules avec du PBS fois. Retirer des cellules nourricières 3T3-J2 par Versène (pré-chauffé à température ambiante) de traitement. Incuber la culture bombé en Versène pendant 5 minutes, puis secouez doucement et aspirer hors des cellules nourricières. </li><li> Laver les cellules souches du follicule pileux soit avec ou PBS Versène une seule fois. </li><li> Ajouter préchauffé (à 37 ° C, critique) trypsine-EDTA (2X) et incuber à 37 ° C pendant 7 minutes ou plus si nécessaire pour les cellules souches du follicule pileux se détacher. Pas plus de 15 minutes est la meilleure. </li><li> Ajouter KCM w / o FEM pour arrêter la trypsine, délicatement la pipette de haut en bas pour disperser les cellules, ralentit au 200g, à 5 minutes. </li><li> Re-suspendre les cellules avec KCM sans EGF, le comte et re-plaque sur la mitomycine C-traitée 3T3-J2 couche de cellules. </li></ol> 4. Immortalisez cellules souches épithéliales Les cellules primaires, cellules souches, même adulte, atteint la sénescence, après passage en série et des mois dans la culture. L&#39;immortalisation des cellules souches primaires est un moyen efficace pour soulager ce problème. <ol><li> Plaque primaires cellules souches épithéliales (~ 350 000 cellules / puits de plaque de 6 également) au passage 1 sur couche nourricière (3T3-J2) et la culture pendant 2 jours. </li><li> Culture PA317 LXSN lignée cellulaire 16E6E7 un jour après l&#39;ensemencement des cellules souches épithéliales primaires avec du DMEM contenant 10% de FBS. (Cette ligne produit le LXSN16E6E7 rétrovirus amphotropes qui encode la E6 et E7 du HPV16 cadres ouverts de lecture, et qui peut être utilisé pour infecter de façon stable et immortaliser de nombreux types cellulaires). </li><li> Traiter PA317 ligne LXSN cellules 16E6E7 à la mitomycine C pendant 2 heures (même protocole avec 3T3-J2 cellules). Ajouter la mitomycine C traités PA317 ligne LXSN cellules 16E6E7 aux cellules souches épithéliales primaires, co-culture pendant 6 jours dans KCM avec EGF. Renouveler KCM frais avec EGF médias tous les 2 jours. </li><li> Retirer 3T3-J2 et PA317 LXSN 16E6E7 cellule avec Versène. Ajouter la mitomycine C cellules traitées PSN 3T3-J2 (néomycine, hygromycine, puromycine résistants, ~ 200 000 cellules par puits) pour les cellules souches épithéliales. Les cellules sont sélectionnés sous 0,2 mg / ml de G418 (Gibco) pour ajouteritional 6 jours. Renouveler frais KCM G418 contenant l&#39;EGF médias tous les 2 jours. Survivre cellules souches épithéliales sera immortalisé les cellules souches. Un puits de cellules souches épithéliales primaires qui ne sont pas co-cultivées avec des cellules PA317 LXSN 16E6E7 doit être défini comme un contrôle pour la sélection au G418, toutes les cellules dans ce puits devrait être tué par G418 après 6 jours de sélection. </li></ol> 5. Les résultats représentatifs Passage précoce des cellules souches épithéliales et la peau immortalisé cellules souches épithéliales forment des colonies serrées constituée de kératinocytes de petite taille (figure 2A) entouré de cellules nourricières. Si cultivées en milieux exempts de sérum avec des suppléments défini, sans une couche nourricière, les cellules souches se dispersent et ne pas former des colonies serrés (ils poussent comme des cellules individuelles et de petits amas). Immortalisé cellules souches épithéliales maintenir un phénotype stable pendant&gt; 12 mois de passage en continu, mais avaient tendance à former des colonies plus serré que les cellules primaires. Ils ne forment pas des colonies indépendantes d&#39;ancrage dans les dosages agar mou, indiquant que ces cellules immortalisées ne possèdent pas les caractéristiques des cellules cancéreuses. Les deux primaires et immortalisées de cellules souches épithéliales expresse follicule pileux marqueur de cellules souches cytokératine 15 (figure 3A), et sont capables de se différencier en follicule pileux épidermique, et les lignages sébacées (figure 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figure 1" /> Figure 1. Pincées poils. Après la cueillette de dispase peau traitée, les follicules pileux apparaissent comme étant soit des poils du club télogène (A) avec une boule de cellules entourant le bas de la chevelure, ou les poils anagène (B) avec un manchon d&#39;épithélium entourant la longueur des cheveux. La région de renflement télogène formes morphologiques distinctes d&#39;une région à la micro-dissection, tandis que le renflement anagène est moins distincte. L&#39;ampoule contient anagène kératinocytes matrice, représentant de transit d&#39;amplification des cellules qui peuvent également être isolées et cultivées. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figure 2" /> Figure 2. Les cellules souches cultures. (A) des cellules souches épithéliales de la peau sous forme serrée colonies épithéliales (désigné par E) lorsqu&#39;elles sont cultivées dans KCM avec une couche nourricière de cellules (désigné par F). (B) explants follicule pileux donnent lieu à des excroissances épithéliales. La région de renflement télogène (désigné par T) est attaché au plat et entouré colonie de cellules épithéliales avec des cellules nourricières. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figure 3" /> Figure 3. Différenciation des cellules souches. (A) Les cellules souches colonies maintenir l&#39;expression des marqueurs épithéliaux tels que la cytokératine 15. (B) des cellules souches épithéliales cutanées peuvent être différenciées selon la lignée follicule pileux tel que déterminé par l&#39;expression K6Hf. (C) Les cellules peuvent être cultivées sur de-epidermalized derme ou d&#39;autres matrices à l&#39;interface air-liquide et sous forme d&#39;un épiderme stratifié avec couche cornée, couche granuleuse et la couche épineuse. (D) Les cellules souches peuvent être induits le long de la lignée sébacées avec l&#39;huile rouge des globules positif.

<ol>
	<li>Jones, P. H., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+human+epidermal+stem+cells+from+transit+amplifying+cells+on+the+basis+of+differences+in+integrin+function+and+expression.">Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.</a> Cell. 73, 713-724 (1993).</li><li>Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E., Albelda, S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C8/144B+monoclonal+antibody+recognizes+cytokeratin+15+and+defines+the+location+of+human+hair+follicle+stem+cells.">The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.</a> J. Cell. Sci. 111, 3179-3188 (1998).</li><li>Bac, S. P., Reneha, A. G., Potte, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+the+intestinal+stem+cell+as+a+paradigm.">Stem cells: the intestinal stem cell as a paradigm.</a> Carcinogenesis. 21, 469-476 (2000).</li><li>Slac, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+epithelial+tissues.">Stem cells in epithelial tissues.</a> Science. 287, 1431-1433 (2000).</li><li>It, M., Li, Y., Yan, Z., Nguye, J., Lian, F., Morri, R. J., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+hair+follicle+bulge+contribute+to+wound+repair+but+not+to+homeostasis+of+the+epidermis.">Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.</a> Nat Med. 11, 1351-134 (2005).</li><li>Spradlin, A., Drummond-Barbos, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Taylo, G., Lehre, M. S., Jense, P. J., Su, T. T., Lavke, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+follicular+stem+cells+in+forming+not+only+the+follicle+but+also+the+epidermis.">Involvement of follicular stem cells in forming not only the follicle but also the epidermis.</a> Cell. 102, 451-461 (2000).</li><li>Oshim, H., Rocha, A., Kedzi, C., Kobayash, K., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+renewal+of+hair+follicles+from+adult+multipotent+stem+cells.">Morphogenesis and renewal of hair follicles from adult multipotent stem cells.</a> Cell. 104, 233-245 (2001).</li><li>Morri, R. J., Li, Y., Marle, L., Yan, Z., Trempu, C., L, S., Li, J. S., Sawick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotsarelis+G+Capturing+and+profiling+adult+hair+follicle+stem+cells.">Cotsarelis G Capturing and profiling adult hair follicle stem cells.</a> Nat. Biotechnol. 22, 411-417 (2004).</li><li>Ro, C., Roch, M., Gu, Z., Photopoulo, C., Ta, Q., Lyle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In vitro Cell. &#38; Dev. Biol. 44, 236-244 (2008).</li><li>Ohyama, M., Vogel, J. C. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=delivery+to+the+hair+follicle.">delivery to the hair follicle.</a> J Investig Dermatol Symp Proc. 8, 204-206 (2003).</li><li>Sugiyama-Nakagiri, Y., Akiyama, M., Shimizu, H. <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+stem+cell-targeted+gene+transfer+and+reconstitution+system.">Hair follicle stem cell-targeted gene transfer and reconstitution system.</a> Gene Ther. 13, 732-737 (2006).</li><li>Stenn, K. S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+the+hair+follicle:+fringe+benefits+of+stem+cell+technology.">Bioengineering the hair follicle: fringe benefits of stem cell technology.</a> Curr Opin Biotechnol. 16, 493-497 (2005).</li><li>Hoeller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+rapid+method+to+construct+skin+equivalents+from+human+hair+follicles+and+fibroblasts.+Exp+Dermatol+10.">An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts. Exp Dermatol 10.</a> , 264-271 (2001).</li><li>Navsaria, H. A., Ojeh, N. O., Moiemen, N., Griffiths, M. A., Frame, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reepithelialization+of+a+full-thickness+burn+from+stem+cells+of+hair+follicles+micrografted+into+a+tissue-engineered+dermal+template+(Integra).">Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (Integra).</a> Plast Reconstr Surg. 113, 978-981 (2004).</li><li>Werni, M., Meissne, A., Forema, R., Brambrin, T., K, M., Hochedlinge, K., Bernstei, B. E., Jaenisch, R. <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+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Par, I. H., Zha, R., Wes, J. A., Yabuuch, A., Hu, H., Inc, T. A., Lero, P. H., Lensc, M. W., Dale, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Kazantsev, A., Goltso, A., Zinchenk, R., Grigorenk, A. P., Abrukov, A. V., Moliak, Y. K., Kirillo, A. G., Gu, Z., Lyl, S., Ginte, E. K., Rogae, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hair+growth+deficiency+is+linked+to+a+genetic+defect+in+the+phospholipase+gene+LIPH.">Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.</a> Science. 314, 982-985 (2006).</li><li>Tola, J., Ishida-Yamamot, A., Riddl, M., McElmurr, R. T., Osbor, M., Xi, L., Lun, T., Slatter, C., Uitt, J., Christian, A. M., Wagne, J. E., Blaza, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+epidermolysis+bullosa+by+transfer+of+wild-type+bone+marrow+cells.">Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.</a> Blood. 113, 1167-1174 (2009).</li><li>Wagne, J. E., Ishida-Yamamot, A., McGrat, J. A., Hordinsk, M., Keen, D. R., Riddl, M. J., Osbor, M. J., Lun, T., Dola, M., Blaza, B. R., Tolar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+transplantation+for+recessive+dystrophic+epidermolysis+bullosa.">Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.</a> N Engl J Med. 363, 629-639 (2010).</li><li>Muraue, E. M., Gach, Y., Grat, I. K., Klausegge, A., Mus, W., Grube, C., Meneguzz, G., Hintne, H., Baue, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Correction+of+Type+VII+Collagen+Expression+in+Dystrophic+Epidermolysis+Bullosa.">Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa.</a> J Invest Dermatol. , (2010).</li><li>Y, H., Kuma, S. M., Kossenko, A. V., Show, L., X, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J Invest Dermatol. 130, 1227-1236 (2010).</li><li>Nishimur, E. K., Grante, S. R., Fishe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+graying:+incomplete+melanocyte+stem+cell+maintenance+in+the+niche.">Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche.</a> Science. 307, 720-724 (2005).</li></ol>

Les méthodes d&#39;extraction de cellule et la culture sont étonnamment faciles décrit et reproductible. Nous avons généré cultures de cellules souches épithéliales de plusieurs dizaines de personnes à travers une large tranche d&#39;âge, y compris les patients avec des défauts de la peau a hérité de 18 ans. Il est préférable de commencer le processus le jour de la récolte des tissus, mais les cellules restent viables dans les médias sur la glace pendant plusieurs jours, ce qui facilite expé...

Les médias des kératinocytes (KCM) [DMEM et Ham s F12 (Gibco, 3:1), l&#39;adénine (Sigma, 180 mm), 10% sérum de veau fœtal (Gibco), la toxine cholérique (CII, 0,1 nM), pénicilline / streptomycine (GIBCO, 100 U / ml et 100 mg / ml, respectivement), de l&#39;hydrocortisone (Sigma, 0,4 mg / ml, 1,1 mM), T/T3 (transferrine, GIBCO, 5 pg / ml, 649 nM, et triiodo-L-thyronine, Sigma, 2 nM) , l&#39;insuline (Sigma, 5 mg / ml, 862 nM), et l&#39;EGF (Sigma, 10 ng / ml, 1,6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

L&#39;homéostasie de tous les tissus d&#39;auto-renouvellement est dépendant sur les cellules souches adultes. Comme les cellules souches indifférenciées subissent des divisions asymétriques, elles génèrent des cellules filles qui gardent le phénotype de cellules souches et de transit d&#39;amplification des cellules (cellules TA) qui migrent de la niche de cellules souches, subissent la prolifération rapide et en phase terminale se différencier pour repeupler le tissu. Les cellules souches épithéliales ont été identifiés dans l&#39;épiderme, du follicule pileux, et de l&#39;intestin que les cellules avec un haut potentiel prolifératif in vitro et en tant que lente vélo étiquette de rétention des cellules in vivo 1-3. Adulte, tissu-spécifique des cellules souches sont responsables de la régénération des tissus dans lesquels ils résident pendant le changement physiologique normale, ainsi que pendant les périodes de stress 4-5. Par ailleurs, les cellules souches sont généralement considérés comme étant multi-puissant, possédant la capacité de donner naissance à plusieurs types de cellules dans le tissu 6. Par exemple, les cellules de rongeurs souches du follicule pileux peut générer l&#39;épiderme, les glandes sébacées et les follicules pileux 7-9. Nous avons montré que les cellules souches provenant de la région bulbe du follicule pileux humain présentent multi-potentialité 10. Les cellules souches sont devenues un outil précieux dans la recherche biomédicale, en raison de leur utilité comme un système in vitro pour étudier la biologie du développement, la différenciation, la tumorigenèse et de leur utilité thérapeutique possible. Il est probable que les cellules souches adultes épithéliales sera utile dans le traitement de maladies telles que dysplasies ectodermiques, monilethrix, Netherton syndrome, la maladie de Menkes, l&#39;épidermolyse bulleuse héréditaire et alopécies 11-13. En outre, les autres problèmes de peau tels que des brûlures, des plaies chroniques et des ulcères bénéficieront de thérapies aux cellules souches 14,15. Etant donné le potentiel de reprogrammation de cellules adultes dans un état ​​pluripotentes (cellules iPS) 16,17, les cellules facilement accessibles et extensible souches adultes dans la peau humaine peut fournir une source précieuse de cellules pour l&#39;induction et le traitement en aval pour un large éventail de maladies, y compris le diabète et la maladie de Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Une façon rapide, robuste d&#39;isoler des cellules adultes viables souches épithéliales de la peau humaine est décrite. La méthode utilise la digestion enzymatique de la matrice collagène de la peau, suivie par la plumaison des follicules pileux et à l&#39;isolement des suspensions de cellules uniques ou fragments de tissus pour la culture cellulaire.

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 ...

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28.2K vues.  University of Massachusetts Medical School. Une façon rapide, robuste d'isoler des cellules adultes viables souches épithéliales de la peau humaine est décrite. La méthode utilise la digestion enzymatique de la matrice collagène de la peau, suivie par la plumaison des follicules pileux et à l'isolement des suspensions de cellules uniques ou fragments de tissus pour la culture cellulaire.

Vidéo: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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 ...

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Biologie

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 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 ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

L&#39;isolement, la culture, et la transplantation de cellules satellites du muscle

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...