Bu çalışma, Ulusal Sağlık Enstitüsü / NCI hibe R01CA-118.916 tarafından finanse edilmektedir

1. İnsan Cilt Epitel Kök Hücreler Özü <ol><li> Epitel kök hücreleri izole işlemine başlamadan önce ilgili medias ve reaktifler (tablo1) hazırlamak gerekiyor. </li><li> Yüz germe işlemleri veya punch biyopsi taze yetişkin insan saçlı deride toplanır, daha sonra DMEM 4 /% 10 FBS / gecede Dispase (4 mg / ml) ° C de inkübe 2-4 saat süreyle inkübasyon 37 ° C de etkilidir. Cilt adet nüfuz enzim için izin maksimum genişliği 1 cm olmalıdır. </li><li> Steril bir Petri kabı içine Transfer cilt, cilt, cilt yüzeyine yakın saç gövdesine açgözlü ve sağlam bir şekilde ve sorunsuz çekerek her saç çekin. Diseksiyon mikroskobu altında kendi morfolojisine göre telojen aşamada follikül seçin, transfer folikülleri (Şekil 1A), 15 ml steril tüpe şişkinliği bölge kesip. Anajen folikülleri 1 / 3 üst folikül kesme, saç folikülü &quot;çıkıntı&quot; bölge (Şekil 1B) elde etmek için de kullanılabilir. </li><li> % 0.05 tripsin-EDTA (Gibco) ve Versene (0.53 mM EDTA 4NA, Gibco) (1:1) karışımı oda sıcaklığında 15-20 dakika süreyle periyodik sallayarak izole folikül parçaları inkübe ekleyebilir, 4 ml DMEM + 10 % FBS 800 rpm&#39;de 5 dakika süreyle durdurmak tepki aşağı spin. Alternatif olarak, folikül doku parçası eksplantlar tek foliküller kültür hücreleri kültür tripsin sindirmek olmadan yerleştirilmiş olabilir. </li><li> Dikkatle supernatant atın (hücreleri kaybetmemek için kaydetmek ~ 0.2-0.5 ml) ve 1 ml EGF olmadan KCM (keratinosit orta), yeniden askıya. </li></ol> 2. Kültür İlköğretim Epitel Kök Hücreler <ol><li> Önce epitel kök hücreler kültür-J2-3T3 hücreleri (besleyici hücreler) aşağıdaki gibi hazırlanmalıdır C-tedavi mitomisin. J2-3T3 hücreleri tedavi öncesi 10% FBS ile DMEM kültür. Kültür ortamı çıkarın ve hücre PBS ile iki kez yıkamak 37 2 saat süreyle serum olmadan DMEM 15μg / ml mitomisin C ° C,% 5 CO 2 hücre tedavisi . Mitomisin C içeren DMEM çıkarın ve PBS ile iki kez yıkayın, hücreler kullanıma hazır. (Mitomisin C-tedavi-J2-3T3 hücreleri de -80 önceden hazırlanmış ve saklanabilir ° C. çözülme ve tohum hücreleri kültür primer epitelyal kök hücreler bir gün önce, 37 inkübe ° C,% 5 CO 2. 150.000-200.000 de ortalama 6-plaka-J2-3T3 hücreleri tavsiye edilir. </li><li> Mitomisin adım 1.5 Tohum izole saç folikülü kök hücre geceleme EGF 37 olmadan KCM 3T3-J2-hücreleri C-tedavi ertesi gün EGF içeren KCM değiştirildi ° C,% 5 CO 2. Hücreler 37 yetiştirilir ° C% 5 CO 2 içeren bir nemli bir atmosfer . Tüm keratinositler KCM içeren EGF ile beslenen her 2 günde bir ve 14-20 gün için yetiştirilmektedir. Mikroskop altında hücre her gün kontrol edin. Saç folikülü kök hücrelerin koloniler (Şekil 2A) oluşturacaktır. Saç folikülü eksplantlar 10-14 gün sonra (Şekil 2B) çıkıntılar oluşturacak. </li></ol> 3. Passage Epitel Kök Hücreler <ol><li> Hücreler PBS ile bir kez yıkayın. Versene (RT önceden ısıtılmış) tedavisi ile 3T3-J2-besleyici hücreler çıkarın. 5 dakika inkübe kültür Versene de bombeli, sonra yavaşça sallamak ve besleyici hücreler aspire. </li><li> Versene veya PBS bir kez ya da kıl folikülü kök hücreler yıkayın. </li><li> Önceden ısıtılmış ekle (37 ° C, kritik) tripsin-EDTA (2X) ve 37 inkübe ° C 7 dakika veya daha uzun bir süre ayırmak için saç folikülü kök hücre için gerekli. Artık az 15 dakika en iyisidir. </li><li> Tripsin durdurmak için KCM w / o EGF ekle yavaşça 200g aşağı dönmeye hücreleri dağıtmak için 5 dakika aşağı yukarı pipet ve. </li><li> Mitomisin C-tedavi 3T3-J2-hücre tabakası EGF, Say ve yeniden plaka olmadan KCM hücreleri yeniden askıya. </li></ol> 4. Epitel Kök Hücreler ölümsüzleştirin İlköğretim hücreleri, hatta erişkin kök hücreler, seri geçit ve kültür ay sonra yaşlanması ulaşır. Birincil kök hücrelerin ölümsüzleşme Bu sorunu gidermek için etkili bir yoldur. <ol><li> Plate 2 gün boyunca birincil epitel kök hücreleri besleyici tabaka geçit 1 (~ 350.000 hücre / 6 plaka) (3T3-J2) ve kültür. </li><li> Kültür PA317 LXSN 16E6E7 hücre hattı DMEM% 10 FBS içeren primer epitelyal kök hücrelerin ekim sonra bir gün. (Bu çizgi HPV16 E6 ve E7 açık okuma çerçeveleri kodlar amphotropic retrovirüs LXSN16E6E7 üretir ve stabil bir şekilde bulaşabilen ve birçok hücre tipleri ölümsüzleştirmek için kullanılabilir). </li><li> PA317 LXSN 16E6E7 hücre hattı 2 saat boyunca mitomisin C (J2-3T3 hücreleri ile aynı protokolü) ile ödüllendirin. Mitomisin C EGF ile KCM 6 gün PA317 LXSN 16E6E7 hücre birincil epitel kök hücreler hattı, ko-kültür tedavi ekleyin. Her 2 günde bir, taze KCM EGF medya yenileyin. </li><li> 3T3-J2 çıkarın ve PA317 LXSN 16E6E7 Versene hücre. Mitomisin-C tedavi 3T3-J2-NHP hücreleri (neomisin, hygromycin, puromycin dayanıklı, iyi başına ~ 200.000 hücre) epitel kök hücreler. Hücreler eklemek için G418 (Gibco) 0.2 mg / ml altında seçilir6 gün itional. EGF medya her 2 günde bir taze G418 içeren KCM yenileyin. Surviving epitel kök hücreler, kök hücreler ölümsüzleştirdi. G418 seçimi için bir kontrol olarak PA317 LXSN 16E6E7 hücre ile birlikte kültür değildir primer epitelyal kök hücrelerin iyi ayarlanması gerekir, bu iyi seçim 6 gün sonra tüm hücreleri G418 tarafından öldürülmüş olmalıdır. </li></ol> 5. Temsilcisi Sonuçlar Deri epitel kök hücre ve epitel kök hücreleri ölümsüzleştirdi erken geçiş, besleyici hücreler ile çevrili küçük keratinositler (Şekil 2A) oluşan sıkı koloniler oluşturur. Besleyici bir tabaka olmadan, tanımlanan takviyeleri ile serum serbest medya kültürü, kök hücreler sıkı koloniler (bunlar tek hücreleri ve küçük kümeler gibi büyümeye) dağıtmak ve formu değil. Ölümsüzleştirilmiştir epitel kök hücreler sürekli geçiş&gt; 12 ay boyunca istikrarlı bir fenotip korumak, ancak birincil hücreler daha sıkı koloniler oluşturmaya eğilimli. Onlar, yumuşak agar tayinlerde bu ölümsüzleştirdi hücrelerin kanserli hücrelerin özelliklere sahip olmadığını belirten ankraj bağımsız koloniler oluşturmak yok. Birincil ve epitel kök hücreleri ölümsüzleştirdi Hem saç folikülü kök hücre belirteci sitokeratin 15 (Şekil 3A) ifade ve epidermal, saç folikülü ve sebase soyları (Şekil 3B-D) ayırt etmek mümkün. <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Şekil 1" /> Şekil 1. kıllar çekti. Toplandıktan sonra dispase ile tedavi edilen cilt, saç folikülleri telojen (B) bir kol ile saç uzunluğu çevreleyen epitel hücrelerinin saç alt çevreleyen bir top ile kulüp kıllar (A) ya da anagen saçlar ya görünür. Telogen çıkıntı bölge anajen çıkıntı daha belirgin iken, mikro-incelemek için ayrı bir morfolojik bölge oluşturur. Anagen ampul de izole ve kültür olabilir transit yükseltme hücreleri temsil eden matris keratinositler içerir. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Şekil 2" /> Şekil 2. Kök hücre kültürleri. (A), cilt hücreleri (F tarafından belirlenmiş) besleyici bir tabaka ile KCM kültür formu sıkı epitel koloniler (E tarafından belirlenmiş) epitelyal kök hücreleri. (B) Saç folikülü eksplantlar epitelyal çıkıntılar doğuran. Telogen çıkıntı bölge (T tarafından belirlenen) çanak bağlı ve besleyici hücreler birlikte epitel hücre kolonisi çevrilidir. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Şekil 3" /> Şekil 3. Kök hücre farklılaşması. (A) Kök hücreler koloniler sitokeratin 15 gibi epitel belirteçleri ifade korumak. (B) Cilt epitel kök hücreler, K6Hf ifade tarafından belirlenen saç folikülü soy boyunca ayırt edilebiliyor. (C), hava-sıvı arayüzü de-epidermalized dermis veya diğer matrisler hücreler kültür ve açığa çıkan tabaka, granüler tabaka ve spinöz tabakası ile tabakalı epidermis oluşturacak. (D) Kök hücreler Oil Red olumlu globülleri yağ soy boyunca teşvik edilebilir.

<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>

Açıklanan hücre çıkarma ve kültür yöntemleri, şaşırtıcı derecede uyduruk ve tekrarlanabilir. Biz, kalıtsal bir cilt kusurları 18 olan hastalar da dahil olmak üzere geniş bir yaş yelpazesinde bireylerin onlarca, epitel kök hücre kültürleri var. Doku hasat gününde işlemine başlamak için en iyisidir, ancak hücreleri gecede nakliye kolaylaştırmak gerekirse, birkaç gün boyunca buz üzerinde medyada canlı kalacaktır. Atılan facelift cilt, tek bir hücre süspansiyonları olarak h?...

Keratinosit medya (KCM) [DMEM ve Ham F12 (Gibco, 3:1), adenin (Sigma, 180 mM),% 10 fetal sığır serumu (Gibco), kolera toksini (ICN, 0.1 nM), penisilin / streptomisin (Gibco, 100 U / ml ve sırasıyla 100 mg / ml), hidrokortizon (Sigma, 0.4 mg / ml, 1.1 mM), T/T3 (transferrin Gibco, 5 mg / ml, 649 nM ve triiodo-l-thyronine, Sigma, 2 nM) , insülin (Sigma, 5 mg / ml, 862 nM) ve EGF (Sigma, 10 ng / ml, 1.6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

Kendini sürekli yenileyen dokuların homeostazı yetişkin kök hücreleri üzerinde bağlıdır. Farklılaşmamış kök hücreleri asimetrik bölünmeler tabi olarak, onlar, kök hücre fenotipi ve kök hücre niş göç, hızlı bir şekilde çoğalması geçmesi ve ölümcül doku yeniden doldurmanız ayırt etmek için transit yükseltme hücreleri (TA hücreleri) korumak kızı hücreler üretir. Epidermis, saç folikülü ve bağırsak epitel kök hücreleri, in vitro proliferatif potansiyeli yüksek ve yavaş bisiklet hücreleri in vivo 1-3 etiket istinat hücreler olarak tespit edilmiştir. Yetişkin, doku spesifik kök hücrelerin, normal fizyolojik ciro sırasında yanı sıra stres 4-5 zamanlarda yaşadığınız dokuların rejenerasyonu için sorumludur . Ayrıca, kök hücreler genellikle 6 doku içinde birden fazla hücre tiplerinin ortaya çıkmasına neden kapasitesine sahip, çok güçlü olması için kabul edilir. Örneğin, kemirgen saç folikülü kök hücreler, epidermis, yağ bezleri, ve saç foliküllerinin 7-9 üretebilir . Biz insan saç folikülü çıkıntı bölge kök hücrelerinin çok-potansiyellik 10 sergilediklerini göstermiştir. Kök hücreler, biyomedikal araştırma, gelişim biyolojisi, farklılaşma, tümörogenez ve onların olası tedavi yarar için eğitim in vitro sistemi olarak yarar nedeniyle değerli bir araç haline gelmiştir. Bu, yetişkin epitel kök hücreleri ektodermal displazileri, monilethrix, Netherton sendromu, Menkes hastalığı, kalıtsal epidermolizis bülloza ve alopesilerde 11-13 gibi hastalıkların tedavisinde yararlı olacağı muhtemeldir . Ayrıca, yanık yaraları, kronik yaralar ve ülser gibi diğer cilt problemleri, terapiler 14,15 ile ilgili kök hücre fayda sağlayacaktır . Pluripotent devlet (iPS hücreleri) 16,17 yetişkin hücreleri yeniden programlama için potansiyel göz önüne alındığında, insan derisi kolaylıkla erişilebilir ve genişletilebilir bir yetişkin kök hücre hastalığı da dahil olmak üzere geniş bir yelpazede hücrelerinin indüksiyonu ve alt tedavisi için değerli bir kaynak sağlayabilir diyabet ve Parkinson hastalığı.

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Insan derisi canlı yetişkin epitelyal kök hücreleri izole bir hızlı, sağlam bir şekilde açıklanmıştır. Bu yöntem cilt kollajen matriks enzimatik sindirim, saç folikülleri ve hücre kültürü için, tek bir hücre süspansiyonları ya da doku parçalarının izolasyonu koparma kullanır.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

JoVE Journal

Biology

28.2K Görüntüleme.  University of Massachusetts Medical School. Insan derisi canlı yetişkin epitelyal kök hücreleri izole bir hızlı, sağlam bir şekilde açıklanmıştır. Bu yöntem cilt kollajen matriks enzimatik sindirim, saç folikülleri ve hücre kültürü için, tek bir hücre süspansiyonları ya da doku parçalarının izolasyonu koparma kullanır.

Video: 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.

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

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

Biyoloji

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.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

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.

İnsan Pankreas Kanseri Xenografts Kök Hücre İzolasyonu

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.

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

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.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

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.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

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.

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

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;

Kas Uydu Hücreleri İzolasyon, Kültür ve Transplantasyon

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.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

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.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

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 ...