Bu çalışma FA Araştırma İttifak ve Arnold Aile Vakfı ve Kök Hücreler ve MD Anderson Kanser Merkezi Gelişimsel Biyoloji Merkezi&#39;nden bir pilot hibe ile desteklendi.

1. Virüs üretim ve iletimi <ol><li> DMEM ortamı (DMEM yüksek glikoz,% 10 FBS inaktive ısı, 2 mM L-glutamin, antibiyotik) 10 ml ~ 7-6 8x10 cm başına 10 plakası bir yoğunlukta plakası Phoenix Ampho hücreleri. 37 gece kuvöz ve kültür Yeri ° C,% 5 CO 2. </li><li> 12, bir vektör kodlama ug ya Oct3 / 4, Sox2, Klf4, c-myc, veya GFP geni (Addgene plazmidler 17.217, 17.218, 17.219, 17.220) ve 35 ul Fugene 6. Kullanılarak sonraki gün transfekte Phoenix hücreleri DMEM ortamı (DMEM yüksek glikoz, 2 mM L-glutamin, hiçbir FBS ve antibiyotikler), 500 ul içinde transfeksiyonuyla karışımı hazırlayın. 20 dakika inkübe edin. Yavaşça% 70-80 konfluent Phoenix hücreleri içeren 10 cm tabak içine DNA kompleksleri Pipeti. </li><li> 8 saat penisilin ve streptomisin içeren DMEM ortamı (DMEM yüksek glikoz,% 10 FBS inaktive ısı, 2 mM L-glutamin) ile birlikte post-transfeksiyon - ortam 6 yerini alır. </li><li> Daha sonra, toplamak virüs içeren 12 medya 4 kez haralıklarla, tüm bölümlerini birleştirmek ve müstakil hücreleri ve enkaz (viral partikülleri içeren ortam da ise dondurulması tavsiye edilmez, bulaşıcı aktivitesini kaybetmeden 2 hafta boyunca buzdolabında muhafaza edilebilir) kaldırmak için filtre 0.45 mikron kullanarak filtrelenmiş. </li><li> Retroviral medya koleksiyonu son gününden önce dondurulmuş bir stok 24 h retroviral iletimi, plaka insan yetişkin fibroblastlarının (pasaj 10, Coriell Laboratories) için. Tohum DMEM yüksek glikoz in oyuk başına yoğunluğu 1x10 5 hücreleri azından 6-de jelatin kaplı levhalar üzerine hücreleri,% 10 FBS, 2 mM L-glutamin, penisilin, streptomisin ve gerekli olmayan amino asitler. </li><li> Phoenix hücrelerden elde edilen viral parçacıklar içeren ortam ile bir sonraki gün ikame DMEM ortamı. Için 1600g azından fibroblast kültürleri ve santrifüj 6-kuyulu plakanın her oyuğuna 6 mg / ml arasında polybrene ile desteklenmiş viral ortamı (1 ml, her Oct4, Sox2, Klf4, ve c-myc ortam, 4 ml toplam) ekleyin 20 de 1 H ° C. Yaklaşık 12 saat after iletimi, fibroblast kültür ortamı ile viral ortamı değiştirin. 24 saat aralıklarla viral enfeksiyon ve santrifüjleme 3 kez gerçekleştirmek. </li><li> Son enfeksiyondan sonra 48 saat DMEM medyada Kültür fibroblastlar. GFP ifade retroviral bir ortam ile paralel transductions ile enfeksiyon kontrol edilmesi. Şekil 1, insan fibroblast santrifüjleme-kolaylaştırılmaktadır retroviral bir enfeksiyon etkinliğini göstermektedir. </li></ol> 2. Yeniden programlama <ol><li> Her sıra jelatin ~ 2x10 5 hücre yoğunluğunda MEF hücreleri tohumlayarak γ-ışınlanmış MEF besleyici katmanları ile 6 oyuklu plakalar hazırlamak fibroblast büyüme ortamında 6 kuyulu plakanın muamele. Ertesi gün fibroblast büyüme ortamında başına iyi ~ yoğunlukta, tripsin / EDTA ve tohum onları 1.2x10 4% 0.05 ile enfekte insan fibroblastları ayrıldı. </li><li> Ertesi gün iEK medya (DMEM/F12,% 20 Nakavt Serum Replacement, non-esansiyel amino asitler, penicilli ile medya yerinen / streptomisin, 2 mM L-glutamin, 0.1 mM β-merkaptoetanol, 20 ng / ml bazal fibroblast büyüme faktörü (bFGF), yeniden programlanması ilk 7 gün süre ile 0.5 mM sodyum butirat ile desteklenmiş. Günlük medya değiştirin. </li><li> Günlük transdük hücrelerinde morfolojik değişiklikler izleyin. HIPS benzeri hücreler kolonileri yaklaşık 5 oluşmaya başlayacak - 10 gün besleyici hücreler üzerine transdük fibroblastlar aktardıktan sonra. HiPSC koloniler yaklaşık 14 izole edilmeye hazır - 28 gün MEF besleyici hücreler bunları kaplama sonra. </li></ol> 3. HiPSC koloniler izolasyonu <ol><li> HiPSC koloniler almadan önce bir gün fibroblast büyüme ortamında γ-ışınlanmış MEF besleyici hücreler (kuyu başına ~ 4x10 4 hücreleri) ile 24-iyi plakaları hazırlamak. Bu aşamada hiPSC koloniler de Matrigel ve mTeSR1 orta kullanarak besleyici özgür kültür transfer edilebilir. </li><li> HiPSC koloniler izolasyon öncesinde fibroblast büyüme Mediu kaldırarak MEF plakaları hazırlamakm ve FBS izlerini kaldırmak için PBS ile MEFS durulama. Daha sonra her oyuğa 7,8 ila 10 uM ROCK inhibitörü Y27632 ile 0.5 ml iEK ortamı (veya Matrigel kuyucuklar durumunda orta mTeSR1) ilave edilir. </li><li> Gerekirse, laminar flow hood (- C Şekil 2A) monte mikroskop altında 21 gauge iğne ile hiPSC kolonileri çevreleyen fibroblastlar kaldırın. PBS ile plakaları durulayın ve Tra-1-81 StainAlive spesifik antikor (1:200, Stemgent) içeren taze iEK medya ekleyin. Sonra 30 dk., 10 uM ROCK inhibitörü ile desteklenmiş taze iEK medya ile antikor içeren medya değiştirin. Floresan mikroskop altında plakaları inceleyin ve mark-Tra 1-81 objektif bir belirteç (Şekil 2B) kullanan pozitif koloniler. </li><li> Şekil 2E ve F. gösterildiği gibi 21 gauge iğne ile birkaç küçük parçalar halinde bir laminer kaputu mikroskop altında Tra-1-81 pozitif hiPSC koloniler, Kes </li><li> Ind içine hiPSC kolonilerinin bir otomatik pipet (P200) transferi parçaları kullanarakMEFS veya Matrigel ile 24-kuyulu plakanın ividual kuyu. Olmayan kalça hücrelerinin transferi kaçının. Bir 37 ° C,% 5 CO2 inkübatöründe plakaları yerleştirin ve hiPSC koloniler 24-36 saat boyunca yapışabilen. </li><li> Değişim iEK veya mTeSR1 (Matrigel yetişen koloniler için) medya günlük. Doğru embriyonik kök benzeri morfolojiye sahip hiPSC koloniler 24 sıra tabağa ilk transferini 48 saat sonra görünür olacaktır. </li><li> El geçit hiPSC koloniler her 6 - 12-iyi ve daha sonra bir 6-iyi plaka üzerine 8 gün. </li><li> Klonal genleşme ve hiPSC hatları kurduktan sonra, Şekil 3 &#39;de gösterildiği gibi immünsitokimya kullanılarak pluripotency belirteçler TRA-1-60, SSEA-4, Oct3 / 4, Sox2 ve Nanog ekspresyon analiz. Genomik bütünlük ve karyotip ve teratom oluşumu analizleri 6,9 kullanılarak elde edilen hatların farklılaşma potansiyeli değerlendirin. </li></ol> 4. Temsilcisi Sonuçlar Retrovirüs içeren medya i ile verimli iletimibaşarılı bir şekilde yeniden programlama için kritik. Bu, Şekil 1 &#39;de gösterildiği gibi etkinliğini izlemek için virüsü her bir deney yeniden programlanması ifade eden bir GFP kullanılarak tamamını transfeksiyon / enfeksiyonu işlemi yürütmek için tavsiye edilir. Tipik olarak 0.5 aralığında olduğu non-konsantre viral ortamı kullanılarak, insan fibroblastlann iletimi yoluyla, 10 içinde açıklandığı gibi GFP eksprese eden virüs titreleri, tespit - 5 x 10 ml (VP / ml) başına 7 viral partiküllerin. Fibroblast son enfeksiyonu sonrası gibi erken 2 gün olarak morfolojisi değiştirin. Tripsinize insan fibroblastları dikkatle MEF besleyici hücreler bunları tohum önce sayılmalıdır. Her hücre hattı farklı büyüme özelliği gösteren ve tohum yoğunluğu yeniden programlama verimliliği için önemlidir çünkü 3 farklı yoğunluklarda (6x10 3, 1.2x10 4, 6 ve plakanın yanı tek başına 2.5x10 4) tohum hücreleri tavsiye edilir. Için kullanılan sodyum butiratİlk 7 - yeniden programlama 14 gün hiPSC formasyonu yaklaşık 5 kat verimliliğini artırır. Sık sık, enfekte fibroblastlar yüksek yoğunlukta ekildi, özellikle fibroblast benzeri hücreler bir kültür çanak sarmak ve Şekil 2A gösterildiği gibi hiPSC koloniler kapsayabilir. Bu durumda, fibroblast tabaka dikkatlice kaldırdı ve hiPSC koloniler (Şekil 2B) ortaya çıkarmak için kaldırılabilir. Daha sonra, hiPSC koloniler iEK ortam ile durulanmış ve Tra-1-81 antikoru ile boyanmış edilmelidir. Fibroblast hücreleri bağlı olarak, yaklaşık 20 - iPSC benzeri morfolojisi ile gösteren koloniler% 40 Tra-1-81 antikoru ile leke yapmaz. 24 - Belirlenen hiPSC koloniler önümüzdeki 12 içinde bir tabak transfer edilebilir. Uzun süreli inkübasyon hiPSCs hızlı bir farklılaşma neden olacaktır. Koloniler elle MEF besleyici hücre veya Matrigel-kaplı levhalar ya üzerine pasajlanmağa devam edilebilir. Genişleme kurulan sonra hiPSC klonlar pluripoten ifadesi için immünsitokimya (ICC) kullanılarak test edilmelidirŞekil 3&#39;te gösterildiği gibi cy markers. Ayrıca, oluşturulan iPS hücre hatları ayrıntılı bir moleküler karakterizasyonu içermelidir: RT-PCR, 9 susturmak transgenlerin bir pluripotency genler ve analizlerin promotorlerde DNA demetilasyon gösterilmesi kullanarak pluripotency gen ekspresyon analizi. <img alt="Şekil 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> Şekil 1.. GFP retroviral medya ile üst üste iki enfeksiyonlarından sonra GFP ifade retrovirüs. (A, B), FA hasta türetilen İnsan yetişkin fibroblast (GM03665, Coriell depo) görüntülenmiştir kullanılarak belirlenir viral iletimi verimliliği. (C, D) İnsan fibroblastlar doğrudan 1600g az 1 saat için 6 oyuklu plakalar üzerine hücrelerinin santrifüj GFP retroviral ortam aynı partiden ile enfekte edilmiştir. Görüntüler enfeksiyondan sonra 48 saat ele geçirildi. <img alt= &quot;Şekil 2&quot; src = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; Şekil 2. HiPSC koloniler izole edilmesi ve belirlenmesi. (A) fibroblastlar çevrili hiPSCs içeren bir tabak Faz kontrastlı görüntü. Hücreler embriyonik kök medyada 21 gün kültüre edildi. Çevreleyen fibroblast tabakası uzaklaştırıldıktan sonra (B, C) aynı hiPSC koloni. (D) Doğru reprogrammed hiPSC koloniler Tra-1-81 yüzey işaretleyici antikor ile canlı boyama ile tespit edilir, steril bir iğne (E, F) kullanarak kesin ve 24 kuyulu plakanın ayrı kuyu aktarılır. <img alt="Şekil 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> Şekil 3. Pluripotency belirli bir belirteç Oct3 / 4, Nanog, Sox2, SSEA4 ve hiPSCs in Tra-1-60 ekspresyonu immünsitokimya ile tespit edildi.

<ol>
	<li>Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Mali, P., Chou, B. K., Yen, J., Ye, Z., Zou, J., Dowey, S., Brodsky, R. A., Ohm, J. E., Yu, W., Baylin, S. B., Yusa, K., Bradley, A., Meyers, D. J., Mukherjee, C., Cole, P. A., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butyrate+greatly+enhances+derivation+of+human+induced+pluripotent+stem+cells+by+promoting+epigenetic+remodeling+and+the+expression+of+pluripotency-associated+genes.">Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.</a> Stem Cells. 28, 713-720 (2011).</li><li>Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, 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>Lowry, W. E., Richter, L., Yachechko, R., Pyle, A. D., Tchieu, J., Sridharan, R., Clark, A. T., Plath, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Dick, E., Matsa, E., Bispham, J., Reza, M., Guglieri, M., Staniforth, A., Watson, S., Kumari, R., Lochmuller, H., Young, L., Darling, D., Denning, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+new+protocols+to+enhance+the+production+and+isolation+of+human+induced+pluripotent+stem+cell+lines.">Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines.</a> Stem Cell Res. 6, 158-167 (2011).</li><li>Ku, S., Soragni, E., Campau, E., Thomas, E. A., Altun, G., Laurent, L. C., Loring, J. F., Napierala, M., Gottesfeld, 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=Friedreich's+ataxia+induced+pluripotent+stem+cells+model+intergenerational+GAATTC+triplet+repeat+instability.">Friedreich's ataxia induced pluripotent stem cells model intergenerational GAATTC triplet repeat instability.</a> Cell Stem Cell. 7, 631-637 (2010).</li><li>Emre, N., Vidal, J. G., Elia, J., O'Connor, E. D., Paramban, R. I., Hefferan, M. P., Navarro, R., Goldberg, D. S., Varki, N. M., Marsala, M., Carson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ROCK+inhibitor+Y-27632+improves+recovery+of+human+embryonic+stem+cells+after+fluorescence-activated+cell+sorting+with+multiple+cell+surface+markers.">The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.</a> PLoS One. 5, e12148-e12148 (2011).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Maherali, N., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Yu, J., Thomson, J. A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Pluripotent+Stem+Cell+Derivation.">Induced Pluripotent Stem Cell Derivation.</a> Essentials of Stem Cell Biology. , 331-337 (2009).</li><li>Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I. I., Thomson, 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=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Woltjen, K., Michael, I. P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung, H. K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells.">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458, 766-770 (2009).</li><li>Zhou, W., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27, 2667-2674 (2009).</li><li>Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., Ebina, W., Mandal, P. K., Smith, Z. D., Meissner, A., Daley, G. Q., Brack, A. S., Collins, J. J., Cowan, C., Schlaeger, T. M., Rossi, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li><li>Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Han, S. S., Williams, L. A., Eggan, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+and+deconstructing+stem+cell+models+of+neurological+disease.">Constructing and deconstructing stem cell models of neurological disease.</a> Neuron. 70, 626-644 (2011).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Özellikle nörolojik ve nörodejeneratif hastalıkların insan, okuyan, özellikle yeterli insan hücresel modelleri erişilememesi nedeniyle zor olmuştur. Farklı hücre tiplerine bunları ayırt etmek için oluşturulan pluripotent kök hücreleri ve potansiyel kolayca elde edilebilen somatik hücreleri yeniden programlamayı yeteneği genetik hastalıkların hücresel modeller oluşturmak için bir olasılık açtı. Ayrıca, iPSCs rejeneratif tıp gelecekte bir çok ümit vericidir. Bu nedenle, pluripotency için ...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10828 </td></tr><tr><td> Non-esansiyel aminoasit </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Sodyum butirat </td><td> Sigma </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81 antikoru </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 antikoru </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> Nanog antikoru </td><td> Hücre Sinyal Teknoloji </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 antikoru </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 antikoru </td><td> Hücre Sinyal Teknoloji </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFS </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Amaç işaretleyici </td><td> Nikon </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCell Teknolojileri </td><td> 05850 </td></tr><tr><td> β-merkaptoetanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polybrene </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Nesne belirteci </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Burada 1-3 sodyum butirat varlığında Oct3 / 4, Sox2, Klf4 ve c-myc (OSKM) kodlayan retroviral vektörlerinin kullanılmasıyla insan kaynaklı hücreler pluripotent kök (hiPSC) içine insan yetişkin fibroblastlar yeniden programlanması bir protokol sunulmuştur. Biz geç geçit yeniden programlamak için bu yöntem kullanılır (&gt; p10), FA hasta türetilen insan yetişkin fibroblastları (GM03665, Coriell depo). Yeniden programlanması yaklaşım, virüs içeren ortam varlığında fibroblastlann tekrarlanan santrifüj kullanılarak yüksek verimli transdüksiyon protokolü içerir. Reprogrammed hiPSC koloniler Tra-1-81 canlı immun kullanılarak belirlendi, pluripotent hücreler bir yüzey belirteci olmayan reprogrammed fibroblastlar ayrılır ve elle 4,5 pasajlanmağa. Bunlar daha sonra hiPSC Matrigel plakaları aktarılır ve doğrudan yeniden programlanması plakadan, besleyici içermeyen koşullar içinde büyütülmüştür. Birinci geçit başlayarak, hiPSC koloniler iEK-l karakteristiği gösterilmektedirike morfolojisi. Bu protokolü kullanmak seçilen kolonilerin fazla% 70 başarıyla genişledi ve hücre hatları içine kurulabilir. Kurulan hiPSC hatları yüzey işaretleyicileri TRA-1-60 ve SSEA-4, hem de Nükleer belirteçler Oct3 / 4, Sox2 ve Nanog dahil olmak üzere karakteristik pluripotency belirteçler gösterilmiştir. Burada sunulan protokolü kurulmuş ve Friedreich ataksisi hastalar ve kontrol bireyler 6, insan yenidoğan fibroblastlar, yanı sıra insan keratinositleri elde yetişkin fibroblastlar kullanılarak test edilmiştir.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

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

Biz retroviral Oct3 / 4, Sox2, Klf4 ve c-myc (OSKM) kodlayan vektörler ve canlı boyama ile doğru yeniden programlanması hiPSC belirlenmesi kullanarak insan kaynaklı pluripotent kök hücreler (hiPSC) Tra-içine insan somatik hücre verimli yeniden programlama için bir protokol sunmak 1-81 antikor.

İnsan İsteyerek Pluripotent Kök Hücre seçme ve ayırma Kolonileri Yetişkin Fibroblastlar gelen reprogrammed

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

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

19.8K Görüntüleme.  University of Texas M.D. Anderson Cancer Center. Biz retroviral Oct3 / 4, Sox2, Klf4 ve c-myc (OSKM) kodlayan vektörler ve canlı boyama ile doğru yeniden programlanması hiPSC belirlenmesi kullanarak insan kaynaklı pluripotent kök hücreler (hiPSC) Tra-içine insan somatik hücre verimli yeniden programlama için bir protokol sunmak 1-81 antikor.

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent İnsan TF lentivirüs İnsan Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi ayarla

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Biyoloji

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.

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

İnsan İsteyerek Pluripotent Kök Hücreler transfekte ve Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCA lentiviral Vektör Kullanma Çevresel Blood İnsan İsteyerek Pluripotent Kök Hücre Üretimi

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

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 ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

İnsan kardiyomiyositleri Üretimi: Besleyici-ücretsiz İnsan Bağlı Pluripotent Kök Hücre A Farklılaşma Protokolü

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Hastadan Elde Edilen Podositleri Oluşturmak için Deri Biyopsilerinin Kullanılması

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Yazar Spotlight: Cilt Biyopsilerinden Hasta Kaynaklı Podosit Üretimi  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Cilt Biyopsilerinden Hasta Kaynaklı Podositlerin Üretimi

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...

Methods Article

İnsan İsteyerek Pluripotent Kök Hücre seçme ve ayırma Kolonileri Yetişkin Fibroblastlar gelen reprogrammed