Bu çalışmalar GJM ve GM NIH UO1HL107443-01 Ödülü tarafından finanse edildi.

1. Periferik kan mononükleer hücreleri (PBMC) izolasyonu ve Genişletme GÜN 0 <ol><li> Sodyum sitrat içeren bir BD Vacutainer CPT Hücre Hazırlanması Tüp içine periferik kan 4 ml çizin. Tüp 8-10 kez ters çevirin ve oda sıcaklığında 30 dakika için 1800 x g&#39;da santrifüjlenir. İdeal olarak, bu adımı toplama 2 saat içinde yapılmalıdır. </li><li> Steril bir 15 ml konik santrifüj tüpüne buffy coat (jel bariyeri ve plazma arasındaki hücre tabakası) pipetleme mononükleer hücre (MH) toplayın. 15 dakika 300 x g santrifüj ile birkaç kez, invert steril fosfat tamponlu salin (PBS) ile 10 ml toplam hacmi getirmek. </li><li> Steril PBS içinde 10 ml içinde tekrar süspansiyon hücreleri ve hücre sayımı ile yerine getirir. 10 dakika boyunca 300 xg&#39;de steril 15 ml konik santrifüj tüpü ve santrifüj içine 1 2x10 6 hücre aktarın. </li><li> Genişleme ortam, 2 ml (EM) (içinde tekrar süspansiyon hücreleri 50 ug / ml içeren Asco QBSF-60 Kök Hücre Ortarbic asit, 50 ng / ml SCF, 10 ng / ml IL-3, 2 U / ml EPO, 40 ng / ml, IGF-1, 1 uM Deksametazon ve 100 ug / ml primocin ya da% 1 pen / strep) ve aktarma bir iyi bir 12-kuyulu plakanın. % 5 CO2 kuluçka makinesi, 37 ° C de hücreler inkübe edin. </li><li> 300 x kalan hücreleri santrifüjleyin % 10 DMSO içeren FBS 10 dk ve donma ~ 2x10 6 hücre / flakon için g. </li><li> PBMC kullanılarak dondurulmuş protokol başlamak için, 10 dakika boyunca 300 xg&#39;de QBSF orta ve santrifüj 10 ml içine hücre 1 vial eritin. EM 2 ml hücreler yeniden süspanse edin ve bir 12-kuyulu plakanın iyi bir transfer. % 5 CO2 kuluçka makinesi, 37 ° C de hücreler inkübe edin. </li></ol> GÜN 3 ve 6. GÜN <ol start="17"><li> Steril bir 15 ml konik tüp hücreleri aktarın ve yapışık hücrelere toplamak QBSF-60 Kök Hücre Orta 1 ml bir kez iyice yıkayın. </li><li> 10 dakika için 300 x g&#39;da hücre santrifüjleyin. </li><li> EM 2 ml hücreler yeniden süspanse edin ve bir 12 de bir transfer-Plaka. % 5 CO2 kuluçka makinesi, 37 ° C de hücreler inkübe edin. </li></ol> 2. STEMCCA Lentivirus ile PBMC&#39;ler ve İletimi GÜN 9 <ol><li> Steril bir 15 ml konik tüp hücreleri aktarın ve yapışık hücrelere toplamak QBSF-60 Kök Hücre Orta 1 ml bir kez iyice yıkayın. </li><li> 10 dakika için 300 x g&#39;da hücre santrifüjleyin. </li><li> 5, ug / polybrene ve STEMCCA lentivirüs ml içeren taze EM, 1 ml hücre yeniden süspanse edin (MOI = 1-10) ve bir 12-kuyulu plakanın de bir transfer. (Lentiviral parçacıklarının üretim tarif protokol bulunabilir <a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a> ) </li><li> 90 dakika için 25 ° C&#39;da 2,250 rpm&#39;de plaka Spin. </li><li> Spin sonra, taze EM ilave 1 ml ekleyin ortam, 2 ml toplam olarak 5 ug / ml polybrene ihtiva eden ve 37 ° C de inkübe plaka,% 5 CO 2 inkübatör. </li></ol> 10. GÜN <ol start="16"><li> Steril bir 15 ml konik tüp hücreleri aktarın ve yapışık hücrelere toplamak QBSF-60 Kök Hücre Orta 1 ml bir kez iyice yıkayın. </li><li> 10 dakika için 300 x g&#39;da hücre santrifüjleyin. </li><li> EM 2 ml hücreler yeniden süspanse edin ve bir 12-kuyulu plakanın 1 iyi transfer. % 5 CO2 kuluçka makinesi, 37 ° C de hücreler inkübe edin. </li></ol> 3. Kaplama MEFS üzerine Hücreleri transdüklü GÜN 11 <ol><li> Coat 2x10 5 hücre az% 0.1 jelatin ve plaka inaktive fare embriyonik fibroblastlar (MEFS) ile / iyi MEF orta (IMDM% 10 FBS,% 1 Temel Olmayan Amino Asitler içeren, 100 mcM β-bir 6-plaka kuyu merkaptoetanol, 2 mM L-glutamin, 100 ug / ml primocin ya da% 1 Pen / Strep.) Infeksiyon başına 3 kuyu hazırlayın. </li></ol> GÜN 12 <ol start="2"><li> Bir steril 15 ml konik tüpe transfer hücreleri<html> ve yapışık hücrelere toplamak QBSF-60 Kök Hücre Orta 1 ml bir kez iyice yıkayın. </li><li> 10 ng / ml bFGF, ve askorbik asit ve EM kullanıldığı gibi aynı konsantrasyonlarda, büyüme faktörü içeren MEF orta 3 ml hücreler yeniden süspanse edin, orta (50 ug / ml Askorbik Asit, 50 ng / ml SCF, 10 ng / ml IL -3, 2 U / ml EPO, 40 ng / ml, IGF-1, 1 uM deksametazon). </li><li> Levha 1 hücreleri ml başına iyi MEFS ihtiva eden bir 6-kuyulu plakanın. / Kuyu ortamı 2.5 ml toplam bFGF, askorbik asit ve büyüme faktörleri ile MEF ortam 1.5 ml ilave edilir. </li><li> 25 500 rpm&#39;de döndürme tabağı ° C&#39;de 30 dakika süre ile. 37 ° C,% 5 CO2 kuluçka makinesi içinde plaka inkübe edin. </li></ol> GÜN 14 <ol start="6"><li> 10 ng / ml bFGF ve 50 mg / ml Askorbik Asit (hiçbir büyüme faktörleri) içeren MEF medyanın 2,5 ml Yem hücrelerini her geçen gün. Aspire ve her besleme ile yüzen hücreler atın. </li><li> Olarak (genellikle haftada bir kez) ihtiyaç duyulan ek MEFS ekleyin. </li></ol>p &quot;&gt; ~ GÜN 20 <ol start="8"><li> Bir kez küçük koloniler yem hücreler insan embriyonik kök hücre (hESC) orta (DMEM/F12 içeren% 20 Knockout Serum Değiştirme,% 1 Temel Olmayan Amino Asitler, 100 mcM β-merkaptoetanol, 2 mM L-Glutamine 2 ml ile günlük, görünür , 100 ug / ml primocin ya da% 1 Pen / Strep ve 10 ng / ml bFGF). </li></ol> 4. Toplama ve IPSC Klonların Genişletme ~ GÜN 30 - 40 <ol><li> 12-plaka inaktive MEFS / iyi hESC orta 1 ml artı Stemolecule Y27632 (ROCK inhibitörü) 10 uM içeren önceden seeded bireysel kuyularda her koloni seçin. </li><li> Yalnızca hESC orta (hayır ROCK inhibitörü) 1 ml bundan sonra günlük hücreleri besleyin. </li></ol> 5. Pluripotency Markers için İmmünofloresan Boyama <ol><li> Boyama Millipore gelen kit &quot;ES Hücre Marker örnek Kiti&quot; kullanılarak ve üreticinin protokolü takip yapıldı. </li></ol> 6. INTEG eksizyonunominal STEMCCA Vektör <ol><li> Kaset yeniden programlanması eksizyonu Kre-IRES-Puro ifade vektörü ve transfeksiyon sonrasında 6 tarif edildiği gibi kısa puromisin seçim kullanılarak elde edilir. </li></ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>Park, I. H., Zhao, R., West, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>Takahashi, K., Tanabe, K., Ohnuki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>Yu, J., Vodyanik, M. A., Smuga-Otto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Stadtfeld, M., Hochedlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

Çıkar çatışması ilan etti.

Burada, yeni toplanmış periferik kan birkaç mililitre izole edilen mononükleer hücreler insan iPSCs oluşturmak için STEMCCA lentiviral vektörünün kullanımı açıklanmaktadır. Protokol ayrıca uzak bir yerde alınan verici hücreleri kullanılarak, dondurulmuş zaman PBMC&#39;ler (buffy coat doğrudan elde), önemli pratik etkileri bir ayrıntı yeniden programlanması için de kullanılabilir. Yeniden programlama indüksiyonundan önce izole PBMC&#39;ler nükleer reprogramming daha makul hücrelerin sağlı...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> BD Vacutainer CPT Hücre Hazırlanması Tüp sodyum sitrat </td><td> BD Biosciences </td><td> 362760 </td><td></td></tr><tr><td> QBSF-60 Kök Hücre Orta </td><td> Kalite Biyolojik </td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td> Atlanta Biyolojik </td><td> S10250 </td><td></td></tr><tr><td> Nakavt Serum Değiştirme </td><td> Invitrogen </td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> ant-am-2 </td><td></td></tr><tr><td> Kalem / Strep </td><td> Invitrogen </td><td> 15140 </td><td></td></tr><tr><td> L-Glutamine </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Temel Olmayan Amino Asitler </td><td> Invitrogen </td><td> 11140 </td><td></td></tr><tr><td> β-merkaptoetanol </td><td> MP Biomedicals </td><td> 190242 </td><td></td></tr><tr><td> Askorbik asit </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> R &amp; D Systems </td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R &amp; D Systems </td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R &amp; D Systems </td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R &amp; D Systems </td><td> 286-EP </td><td></td></tr><tr><td> Deksametazon </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> Polybrene </td><td> Sigma </td><td> H-9268 </td><td></td></tr><tr><td> bFGF </td><td> R &amp; D Systems </td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES Hücre Marker Numune Seti </td><td> Millipore </td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

Dört transkripsiyon faktörleri, Oct4, Klf4 ve ektopik ekspresyonu ile, Sox2 ve cMyc, insan somatik hücre sözde indüklenmiş pluripotent kök hücreleri (iPSCs) 1-4 üreten, bir pluripotent durum için dönüştürülebilir. Hastaya özel iPSCs embriyonik kök hücreler (EKH) çevreleyen ve olası immün red baypas edecek etik kaygılar yoksundur. Böylece, iPSCs hastalığı modelleme çalışmaları, farmakolojik bileşikler tarama ve rejeneratif tedaviler 5 için oldukça dikkat çekmiştir. Bu Yamanaka faktörler 6 kodlayan bir tek vergilendirilebilir polisistronik lentiviral Kök Hücre Kaseti (STEMCCA) kullanılarak farklı akciğer hastalıkları olan hastalardan alınan transgeni içermeyen insan iPSCs üretimi göstermiştir. Bu IPSC çizgiler deri fibroblastları, yeniden programlanması için en yaygın olarak kullanılan hücre tipinden elde edildi. Normal olarak, fibroblastlar elde hücrenin genişlemesi punch biyopsi ardından bir cilt gerektirirBirkaç pasajlar için kültür s. Önemli olarak, grup halinde iPSCs 7-9, insan periferik kan hücrelerinin yeniden programlanması bildirilmiştir. Bir çalışmada, STEMCCA vektörünün bir Tet indüklenebilir versiyon kan hücreleri eşzamanlı olarak ters tetrasiklin Transaktivatörü kodlayan bir yapısal olarak aktif lentivirüs ile enfekte edilmesi için gerekli olan, 9 kullanılmıştır. Fibroblastlar tersine, periferik kan hücrelerinin büyük ölçüde hastanın rahatsızlık ve sıkıntı azaltarak, minimal invazif yoluyla toplanabilir. Kurucu bir tek vergiye tabi vektör kullanarak kan hücreleri yeniden programlama için basit ve etkili bir protokol daha geniş bir araştırma toplum için erişilebilir kılarak IPSC teknolojisi uygulama hızlandırabilir. Bundan başka, periferal kan hücrelerinin yeniden programlanması deri biyopsileri kaçınılmalıdır (örn. Anormal yara) ya da bağlı olarak önceden var olan hastalık durumlannın preventi için gereken bireyler dan iPSCs üretilmesine olanak tanırpunch biyopsi için ng erişim. Burada yapısal 4 faktörleri ifade eden bir tek floxed-vergiye tabi lentiviral vektörü kullanılarak periferik kan mononükleer hücreleri (PBMC) insan iPSCs üretimi için bir protokol göstermektedir. Yeni toplanmış ve çözülmüş olan PBMC STEMCCA lentivirüs ile transdük önce olduğu gibi ortam içeren askorbik asit, 10,11 tarif 9 gün, SCF, IGF-1, IL-3 ve EPO için genişletilir. Hücreler daha sonra MEFS üzerine kaplanır ve ESC gibi koloniler iki hafta sonra enfeksiyon canlandırılabilir. Son olarak, seçilen klonlar genişletildi ve pluripotency işaretleri SSEA-4, Tra-1-60 ve Tra-1-81 ekspresyonu açısından test edilmiştir. Bu protokol kan kolayca erişilebilir 4 ml insan iPSCs üretimi için güvenilir bir yöntem sağlayan, basit, güçlü ve son derece tutarlıdır.

Tek bir lentiviral vektörü kullanılarak, insan PBMC&#39;leri iPSCs üretimi için basit ve etkili bir protokol göstermektedir. Şekil 1A protokol şematik bir temsilini gösterir. Kan BD Vacutainer CPT Hücre Hazırlanması Tüp sodyum sitrat ile toplanır ve santrifüj sonrasında, mononükleer hücreler polyester jel ve plazma (buffy coat) (Şekil 1B) arasındaki arayüz alınabilir. İzole PBMC&#39;ler sonra 9 gün boyunca kültürü yayılmaktadır. Şekil 2 hü...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

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

Burada tek bir lentiviral yeniden programlanması vektörü kullanılarak periferal kan 3-4 ml insan iPSCs üretimi için basit ve etkili bir protokol göstermektedir. Hazır kan hücrelerinin yeniden programlanması daha geniş bir araştırma toplum için erişilebilir kılarak IPSC teknoloji kullanımı hızlandırmak için vaat ediyor.

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, ...

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Biology

26.4K Görüntüleme.  Boston University School of Medicine. Burada tek bir lentiviral yeniden programlanması vektörü kullanılarak periferal kan 3-4 ml insan iPSCs üretimi için basit ve etkili bir protokol göstermektedir. Hazır kan hücrelerinin yeniden programlanması daha geniş bir araştırma toplum için erişilebilir kılarak IPSC teknoloji kullanımı hızlandırmak için vaat ediyor.

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Dört Transkripsiyon Faktör Fare Embriyonik Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi, Doksisiklin lentiviral İletim Sistemi Đndüklenebilir

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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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 ...

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 ...

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

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

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

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 ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP Retroviral Vector kullanarak İsteyerek Pluripotent Kök Hücrelerinin (iPSCs) içine İnsan somatik hücreleri yeniden programlama

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Bir Multititre Plaka Biçimi İnsan Pluripotent Kök Hücreler Nöron Hızlı ve Verimli Üretim

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.