We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

Not:. Bu yöntem, Awe ark 10&#39;da rapor araştırmada kullanılmıştır. STEMCCA 1. yeniden programlanması Yetişkin insan dermal fibroblastlarında <ol><li> Çözülme yetişkin dermal insan fibroblastlarının (HUFS), 2 dakika boyunca 37 ° C su banyosu içinde geçiş 4 ya da daha düşük, su ile şişenin üst batırmak için özen. </li><li> 15 ml konik bir şişe, insan fibroblast 1 ml&#39;lik bir bulamaç koyun ve yavaş yavaş damla damla bir tarzda, 37 ° C&#39;ye önceden ısıtılmış, standart HUF ortam, 4 ml yerleştirmek, dimetil sülfoksit (DMSO) sulandırmak ve yeniden askıya hücreleri. </li><li> Oda sıcaklığında 10 dakika boyunca 200 x g&#39;de şişe santrifüjleyin. Süpernatan aspire ve / mi 100,000 hücre süspansiyonu elde etmek üzere ortam yeterli bir hacimde hücrelerin yeniden askıya ve daha sonra, altı gözlü bir plakanın bir kuyuya 2 mi ekleme,% 0.2 jelatin ile 20 dakika için önceden kaplanmış. NOT: hat ve durumun başına hücre eşit sayıda iyi bir kardeş fo seribaşı gerekiyorr hücre sayımı / enfeksiyon (İB) hesaplama çokluğu. </li><li> Eşit hücreleri dağıtmak için ileri geri yan ve plaka yan Kaya. Nemlendirilmiş bir kuluçka makinesi içinde 37 ° C&#39;de,% 5 CO2 seviyesinde bir gece boyunca inkübe edin. </li><li> Transdüksiyon gününde hücre kardeş gelen sayısı (ler) de, ilk HUF sonra kaplama (lar) ile, takriben 24 saat elde edin. </li><li> 2 x 10 8 TU, ya da eşdeğeri, lentiviral konsantre (STEMCCA) çözülme ve HUF medya 1 x 10 8 TU / ml sulandırmak. </li><li> 8 ug / ml transfeksiyon maddesi ile takviye edilmiş HUF ortam içinde 10-İB oranında hücreleri nakletmek. Eşit kalıt kuyu karıştırın ve nemlendirilmiş bir kuluçka makinesi içinde 37 ° C&#39;de,% 5 CO2 seviyesinde bir gece boyunca inkübe edilir. </li><li> Lentiviral transdüksiyon sonra yaklaşık 24 saat, standart insan pluripotent kök hücre (PSC) orta HUF medya geçiş. </li><li> Gün 2 6 aracılığıyla, insan PSC orta günlük ortamı değiştirmek. </li><li> 6. günde, iki% 0.2 jelatin kaplı 10 cm plakayıHUF ortam 12 CF1 fareler elde edilen 1,5 ila 10 x 1.75 ila 6 MEF&#39;ler. </li><li> Günde 7 günü, dikkatli olmak arasında eşit tüm hücreleri kaplama ile, bir 1:16 oranında altı plaka ve geçit bir kuyudan günde 7 yeniden programlanması fibroblastlar kaldırın 100&#39;den fazla xg, kullanım oda sıcaklığı tripsin santrifüj değil bir gün önce MEF&#39;ler ile kaplandı taze insan PSC medya, iki 10 cm tabak,. 37 ° C bir saat yönünde sarmal hareket ve yer hücre toplulukları dağıtmak,% 5 CO2, gece boyunca inkübatör nemlendirilmiş. </li><li> Yaklaşık 48 saat sonra MEF&#39;ler ve her gün üzerine yeniden programlanması hücreleri tohumlama sonra, taze medya ile harcanan insan PSC medya dışarı değiştirmek. </li><li> 3-4 hafta sonra, bir 21 gauge iğne ile morfoloji gibi tipik ESC ile ayrı ayrı koloniler, çekme ve% 0.2 jelatin kaplı tek tek oyuklara belirli kolonilerin yaklaşık 8-10 bireysel parçaları yerleştirilerek 24 kuyucuklu plaka içerisine alt klonlamak 24-iyi plaka ön-çekirdekİnsan PSC ortamda CF1 fareler türetilen MEF&#39;ler ile ed. NOT: seçilmiş kolonilerin büyütüldükten sonra, koloniler protein seviyesinde embriyoit vücut formasyonu ve Pluripotency markerlerinin ifadesi analizi ile pluripotent, özelliği gerekir. </li></ol> 2. Vektör Entegrasyon Site Analizi ve STEMCCA eksizyonu <ol><li> 10 anlatıldığı gibi olmayan kısıtlayıcı doğrusal amplifikasyon PCR ile STEMCCA entegrasyon siteyi analiz. </li><li> Eski insan ESC orta kapalı aspire genomu, tüketim STEMCCA istenen alana bir entegrasyon sağlamak için tahlil sonra. Daha sonra, 24 saat süre ile standart PSC ortam 3 ml 8 ug / ml transfeksiyon maddesi ile konsantre Adeno Cre PuroR virüsünün 45 ul birleştirir. </li><li> 24 saatlik bir bekletme süresinden sonra, karışık bir viral süpernatan aspire ve insan PSC ortamı ile iki kez yıkama hücreleri. 5 gün arasında bir süre için 2 ug / ml puromisin ile taze insan PSC ortamlarını ortam günlük wit değiştirmekh taze medya ve antibiyotik. </li><li> 5 gün sonra, alt-klon 12-oyuklu bir plaka içerisinde,% 0.2 jelatin kaplı oyuklara üzerine 21-gauge iğne ile koloniler kalan CF1mice türetilen MEF&#39;ler ile önceden kaplanmış ve yukarıda açıklandığı gibi genişletmek. Not: yeterli genişleme donmuş stok elde etmek üzere sonra besleyici içermeyen koşullar dönüştürme gereklidir. En hücreler başarılı bir şekilde transdüksiyona olmayacak ve kuluçkalama 5 günlük bir süre boyunca antibiyotiğe ölmek olarak en az% 95, kök hücre koloni ölümü bekliyoruz. Adeno Cre PuroR faktörü içermeyen koloni örnek reexposing, enfekte olmuş hücrelerin konukçu kromozomları içine 0.001-1 verimliliği% entegre olmasına rağmen, entegrasyon 13 oluştu doğrular% 100 hücre ölümünü sağlamak için puromisin için. </li><li> Matris bir sıvı olduğu kadar, yaklaşık 2 saat (imalatçının önerileri) bir süre boyunca buz üzerinde ön aliquoted taban membran matrisi bir vial çözülme ve soğuk bazal ortam içinde 1:30 arasında nihai bir konsantrasyona kadar sulandırmak. Cooyuk başına 1 ml bir altı yuvalı plaka içindeki yuvalara istenen sayıda. 1 saat oda sıcaklığında bekletin. </li><li> Bir de daha önce MEF&#39;ler ile kaplanmış 30 koloniler en az 20 (18 iğne, ya da bir cam veya plastik &quot;ucu&quot; kullanarak) çapraz çizgili. MEF canlandıran azaltmak ve plaka aktarmak dikkatli olun. <ol><li> Steril plastik pipet basit kullanımına açık alev üzerinde, daha karmaşık ısıtma, farklı yaklaşımların bir numara ile bir cam Pasteur pipet çekerek ve terbiye çapraz tarama cihazı üretmek ya, bizim olduğu gibi, 21- ve / veya 18 gauge iğne. </li></ol></li><li> Kaplanmış de sonra inkübasyon matrisi aspire. </li><li> 200 ul pipet ile eski plaka hafif bir kazıma ile kolonilerin parçasını çıkarın ve dikkatli bir şekilde matris kaplı plaka taze kombine ortam 1, 3 ml içine koyun. 37 ° C,% 5 CO2 ile nemlendirilmiş kuluçka makinesi içinde bir gece boyunca inkübe edin. Değişim Medya 48 saat Pasajlanması sonra. </li><li> Ticari DNA ekstraksiyon kitleri kullanılarak,% 80&#39;den fazla confluency, eksize-sonrası ve besleyici içermeyen dönüştürülen hiPSCs genomik DNA ayıklayın. </li><li> STEMCCA lentivirüs eksojen entegrasyon için özel primerler ile birlikte uygun bir eksizyon için Analiz: gDNA-Hendo-MycS ileri, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA-hWPRE-ters, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. 10-uM stok solüsyonu PCR-dereceli su ile Liyofilize primerler sulandırın. </li><li> Aşağıdaki beş aşamalı protokol ile bir PCR çalıştırın: ilk denatürasyon 3 dakika boyunca 95 ° C&#39;de; 15 saniye için 72 ° C &#39;de 15 saniye ve uzatılması için 62 ° C&#39;de 20 saniye, primer tavlama için 98 ° C&#39; de denatürasyon, aşağıdaki her bir 35 döngü 3 dakika boyunca 72 ° C &#39;de, tek bir döngü son uzatma eklenmiştir. </li><li> Agaroz 1.5 gram tartılması ve 50 mi 1 x Tris-asetat-EDTA tampon içine yerleştirerek bir% 3 agaroz jel olun. Agaroz kadar Mikrodalga çözülmüş ve DNA jel leke olarak yer p de olduğuroper seyreltme, karıştırma, ve oda sıcaklığında 1 saat boyunca katılaşmaya jel kasetine doğru hacminin dağıtımı. </li><li> PCR ürününün yüklenebilir 12 ul% 3 agaroz jel içine yükleme boyası 3 ul ile karıştırılır. 80 V de 30 dakika boyunca jel başlat </li><li> Uygun eksizyon gösteren bir grubun olmaması için ultraviyole ışık ile gözünüzde canlandırın. </li></ol> Öncesi ve Sonrası-kesilmiş hiPSCs gelen Kantitatif PCR kullanarak Gen İfadesi Farklar 3. kantitasyonu <ol><li> Ticari RNA ekstraksiyon kitleri kullanılarak kesilmiştir-sonrası ve besleyici içermeyen dönüştürülen hiPSCs toplam RNA ayıklayın. </li><li> -Ters uyarlamak üreticinin talimatlarına uygun olarak ticari kitler kullanılarak toplam RNA 1 ug kadar. Demirli-oligo (dT) 18 ve rasgele heksamer primer kullanın. NOT: PCR protokolü 4 kb daha fazla veya daha az gen transkriptleri için özel olduğundan emin olun. Spesifik primerler hangisi gen STEMCCA başlangıçta entegre i için yapılan gerekecektirnGoogle&#39;a. </li><li> 20 mcM stok solüsyonu PCR-dereceli su ile liyofilize astar sulandırın. </li><li> 10 mcM UPL prob oluşan reaksiyon 20 ul, 2x LightCycler 480 Problar Lisans başına numune 5 ng kullanın ve ileri 20 uM ve geri primerler. </li></ol> GMP dereceli Koşullar içine 4. Dönüşüm <ol><li> GMP dereceli koşullarına üzerinde post-eksize hiPSCs geçiş ilk gününde, kombine insan PSC oluşan medya karışımı yapmak ortam 1 (kombine medya 1) ve klinik notu iyi üretim uygulamaları (GMP) uyumlu medya (kombine medya 2 ) 80:20 oranında. </li><li> Aspire eski kombine medya 1 ve 80:20 oranında yeni kombine medya 1 ve 2, önceden ısıtılmış 3 ml, koyun kapalı. Bu özel oran ile 3 gün boyunca günde ortamı değiştirin. Lütfen 37 ° C,% 5 CO2 inkübatör içine yerleştirin hücreleri. </li><li> 4. günde, 50:50 oranında bir araya ortamı 1 ve 2 olun. Eski orta ve Replac kapalı aspire50:50 oranında bir araya ortam 1 ve 2 ile önceden ısıtılmış ortam, e. Bu özel oran ile 3 gün boyunca medya değiştirin. Lütfen 37 ° C,% 5 CO2 inkübatör içine yerleştirin hücreleri. </li><li> 7. günde, bir 20:80 oranında bir araya ortamı 1 ve 2 olun. Aspire eski orta kapalı ve 20:80 oranında kombine medya 1 ve 2 ile, önceden ısıtılmış medya ile değiştirin. Bu özel oran ile 3 gün boyunca medya değiştirin. Lütfen 37 ° C,% 5 CO2 inkübatör içine yerleştirin hücreleri. </li><li> 100 oranında: günde 10, bir 0 kombine Ortam 1 ve 2 yapmak. Aspire eski orta kapalı ve 0 kombine medya 1 ve 2 ile, önceden ısıtılmış medya ile değiştirin: 100 oranında. Bu özel bir oranda her gün medya değiştirin. Hücreler, tam olarak tanımlanmış kseno-içermeyen ortam artık. Lütfen 37 ° C,% 5 CO2 inkübatör içine yerleştirin hücreleri. Not: Bu, tüm dönüştürme işlemi sırasında bir 18 gauge iğne ile rutin pasajı uygun koloni büyüklüğü ve yoğunluğu sağlamak için gereklidir. S Planıkoloni boyutu akma ve farklılaşma olarak her 4-5 gün hücreler assaging. </li><li> Üretici tarafından tavsiye edildiği gibi tanımlanmış bir kseno-serbest alt-tabaka ile, altı gözlü bir plakanın üzerindeki çukurların kaplayın on. </li><li> Mekanik geçit konfluent iyi, zaten 18-gauge iğne ile altı-kuyu plaka, kseno ücretsiz medya koşullarını dönüştürülür. Önemlisi, medya pre-condition için pasaj önce, 1 saat 1x ROCK inhibitörü olan hücreler üzerinde yeni medya (kombine medya 2) yerleştirin. </li><li> Hücreler transfer ediliyor, yeni plaka kapalı sentetik substrat solüsyonu aspire. Eski plaka kök hücre kümeleri içeren tüm ortamları çıkarın ve yeni plaka transfer. </li><li> (Kez inkübatör içinde, doğrudan herhangi bir şekilde dokunulmaz olan ideal plakaları) geri az fiziksel rahatsızlık ile 2 gün boyunca 37 ° C,% 5 CO 2 kuvöz içine yerleştirin hücreleri maksimum eki izin vermek. NOT: Hücreler günlük medya değişiklikleri gerektirir. Sürekli passaging her 4 günde önemli olacaktır. 21-gauge iğne, optimum ESC-benzeri morfolojisi (yüksek Nükleositoplazmik oranı) ile kolonilerin geçişi yalnızca parçalarını kullanarak. Bu iyi büyür ve sonunda homojen koloniler verecek uygun hiPSC koloniler için seçimi sağlayacaktır. Kolonilerin gibi ESC derivasyon genel zaman ilk kombine medya değişiminden sonra 15-20 gün arasındadır. </li><li> 18-gauge iğne ile ilk çapraz-hatch ile kolonilerin tüm GMP dereceli sonrası eksize hiPSCs aşağı dondurun. 200 ul pipet kullanılarak atık tüm parçaları kaldırın ve 4 ° C&#39;de 5 dakika süreyle 200 x g&#39;de 15 ml konik bir şişe ve santrifüj hücreleri ihtiva eden tüm medya aktarın. </li><li> Hücreler santrifüje tabi olsa da, soğuk kombine ortam 2 eşit hacimlerde oluşan ve% 7.5 nihai bir DMSO konsantrasyonu ile orta donması dondurma ortamı olun. </li><li> Hücreler pelet sonra, eski orta aspire ve dondurma medya ile pelet yeniden askıya-damla damla. Hemen trdonma şişelere ansfer ve ° C derin dondurucuda bir -80 koydu. </li></ol> 5. karakterizasyon GMP dereceli Post-eksize hiPSCs <ol><li> 3. adımda qPCR yöntemleri de belirtildiği gibi takip anahtar Pluripotency belirteçlerinin (Sox2 Oct4 ve Nanog) aynı adımları Tablo 1&#39;de listelenen primerler kullanılarak ifadesinin miktarını belirlemek için, QPCR kullanarak Pluripotency belirteçlerinin sonrası dönüşüm uygun bir ifade sağlamak için Aşama 3 &#39;de gösterildiği gibi, aynı adım. </li><li> Kullanımlar, daha önce 10 yayınlanan kitte yer alan standart koşullara uygun olarak, insan olmayan bir sialik asit Neu5Gc (N -glycolylneuraminic asit) tespit etmek için akış sitometrisi. </li><li> Yavaşça 1x Fosfat tamponlu serum fizyolojik (PBS) kullanarak, hücrelerin bulunduğu hücre kültürü plakaları yıkayın. </li><li> Oda sıcaklığında 5 dakika boyunca 1x ayrışma ajanı kullanılarak koloni ayrıştırmaları. </li><li> Aşama 5.4, 5-dakikalık inkübasyon süresi esnasında verilen engelleme çözeltisi (hazırlamakbuz soğukluğunda PBS içinde bloke edici madde, bir% 0.5 yaparak Kit) birleştirildi. </li><li> Flow sitometri analizi için tüplerin aşağıdaki kümesi Etiket: lekesiz; 4 &#39;, 6-diamidino-2-fenilindol (DAPI) DAPI; Kontrol antikoru 1: 200; Birincil antikor, 1: 200; İkincil antikor, 1: 200 Eşek anti-tavşan IgG&#39;si (H + L); Örnek MEF&#39;ler; Matris üzerinde örnek sonrası eksize hücreler; ve Numune sentetik alt tabaka üzerine hücreleri sonrası çıkarıldı. </li><li> Yavaşça, 15 ml konik bir tüp çözeltisi ve aktarma içeriği bloke 2 ml ilave adım 5.4 hücre agregatları neden olacaktır. 4 ° C&#39;de 5 dakika boyunca 80 x g&#39;de santrifüjleyin. </li><li> Adım 5.7 belirtildiği gibi çözüm ve santrifüj engelleme ile iki kez hücreleri yıkayın. </li><li> Yavaşça her bir antikorun uygun seyrelme hacmi ile bloke etme çözeltisi 100 ul, yaklaşık 1 x 10 6 hücre yeniden askıya. Yumuşak, 1 saat boyunca 4 ° C&#39;de sallanarak inkübe edin. </li><li> Adım 5.8 gibi yıkar üç kez tekrarlayın. </li><li> Seyreltici Bu yüklenmiştir 400 ul hücre pelletini yeniden askıyaffer 1 ile (kit): tüpler 2, 6, 7 için DAPI 100 ve 8 adımda 5.6 altında listelenen. </li><li> 40 mcM süzgecinden Gerinim hücreleri ve akış sitometresinin koşuyoruz. </li></ol>

<ol>
	<li>Sommer, C. A., Mostoslavsky, 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+evolving+field+of+induced+pluripotency:+Recent+progress+and+future+challenges.">The evolving field of induced pluripotency: Recent progress and future challenges.</a> J Cell Physiol. 228, 267-275 (2013).</li><li>Lu, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+xeno-free+and+feeder-free+culture+system+for+the+derivation,+expansion+and+direct+differentiation+of+transgene-free+patient-specific+induced+pluripotent+stem+cells.">A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells.</a> Biomaterials. 35, 2816-2826 (2014).</li><li>Karumbayaram, S., 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=From+skin+biopsy+to+neurons+through+a+pluripotent+intermediate+under+Good+Manufacturing+Practice+protocols.">From skin biopsy to neurons through a pluripotent intermediate under Good Manufacturing Practice protocols.</a> Stem Cells Transl Med. 1, 36-43 (2012).</li><li>Mostoslavsky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+magic+act+of+generating+induced+pluripotent+stem+cells:+many+rabbits+in+the+hat.">Concise review: the magic act of generating induced pluripotent stem cells: many rabbits in the hat.</a> Stem Cells. 30, 28-32 (2012).</li><li>Takahashi, 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=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>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=Induced+pluripotent+stem+cell+generation+using+a+single+lentiviral+stem+cell+cassette.">Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.</a> Stem Cells. 27, 543-549 (2009).</li><li>Somers, 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>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=Excision+of+reprogramming+transgenes+improves+the+differentiation+potential+of+iPS+cells+generated+with+a+single+excisable+vector.">Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.</a> Stem Cells. 28, 64-74 (2010).</li><li>Papapetrou, E. P., 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=Genomic+safe+harbors+permit+high+beta-globin+transgene+expression+in+thalassemia+induced+pluripotent+stem+cells.">Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells.</a> Nat Biotechnol. 29, 73-78 (2011).</li><li>Awe, J. P., 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+and+characterization+of+transgene-free+human+induced+pluripotent+stem+cells+and+conversion+to+putative+clinical-grade+status.">Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.</a> Stem Cell Res Ther. 4, (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receives+FDA+Clearance+to+Begin+World's+First+Human+Clinical+Trial+of+Embryonic+Stem+Cell-Based+Therapy.">Receives FDA Clearance to Begin World's First Human Clinical Trial of Embryonic Stem Cell-Based Therapy.</a> Geron Corporation Press Release. , (2009).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. 64, 3810-383791 (2012).</li><li>Mitani, K., Kubo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+as+an+integrating+vector.">Adenovirus as an integrating vector.</a> Curr Gene Ther. 2, 135-144 (2002).</li><li>Patterson, 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=Defining+the+nature+of+human+pluripotent+stem+cell+progeny.">Defining the nature of human pluripotent stem cell progeny.</a> Cell Res. 22, 178-193 (2012).</li><li>Schroder, A. 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=HIV-1+integration+in+the+human+genome+favors+active+genes+and+local+hotspots.">HIV-1 integration in the human genome favors active genes and local hotspots.</a> Cell. 110, 521-529 (2002).</li><li>Hockemeyer, D., 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=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nat Biotechnol. 29, 731-734 (2011).</li></ol>

James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

Biz faktör ücretsiz hiPSCs kaynaklanan ve gelecekteki insan tedavide mansap hücre farklılaşması için GMP dereceli koşulları içine bu hücrelerin dönüştürerek onları klinik açıdan yapma bir metodoloji açıklar. Bu protokol, hücre tiplerinde çeşitli geniş çapta uygulanabilir olsa da, bağlı hastadan çıkarma kolaylığı ve kişiye özel insan terapötiklere uygulanabilirlikleri, insan dermal fibroblastları yeniden programlamak seçtik. Sınırlamalar klinik açıdan anlamlı hücre türevleri i?...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

İnsan uyarılmış pluripotent kök hücreler (hiPSCs) lentiviral bazlı yeniden programlama yöntemleri ile üretilebilir. Bununla birlikte, genomun aktif transkribe bölgelerde kalan potansiyel onkojenik genlerin izleri, insanlar için terapötik uygulamalar 1 kullanım için potansiyelini sınırlamaktadır. Buna ek olarak, tedavi edici olarak uygun türevleri olarak, kök hücre yeniden programlanması ya da farklılaşma elde edilen insan olmayan antijenleri, insan klinik bağlamda 2&#39;de kullanılan bu hiPSCs engellemektedir. Bu videoda, biz yeniden programlanması ve eksojen transjenlerin ücretsiz faktör ücretsiz hiPSCs analiz etmek için bir prosedür sunuyoruz. Bu hiPSCs sonra lentivirüs ihtiva eden özel bir intron gen sentezleme anormallikleri analiz edilebilir. Bu analiz, önceden gen ekspresyonu farklılıklarını 3 tespit etmek için kullanılan daha hassas teknikler üzerinde bir avantaja sahiptir hassas bir kantitatif polimeraz zincir reaksiyonu (PCR) kullanılarak gerçekleştirilebilir. Içine tam dönüşümKlinik dereceli iyi üretim uygulamaları (GMP) koşullar, insan klinik önemi veriyor. Bizim protokol sunan başka bir metodoloji sağlanan akım güvenli liman kriterleri genişletmek ve insan terapötik uygulamalar-nedeniyle insan olmayan antijenlere karşı herhangi bir immünojenisite riskini ortadan kaldırmak gerekir GMP dereceli hiPSCs, türetmek için faktör içermeyen karakterize hiPSC-tabanlı türevlerini içerir ki. Bu protokol, her tür lentiviral yeniden programlanması hücrelerin geniş çapta uygulanabilir GMP dereceli koşullarına programlanması hücreleri dönüştürmek için yeniden üretilebilir bir yöntem sağlar.

Bu STEMCCA Lentiviral bazlı yeniden programlama yaklaşımı kullanarak, klinik sınıf faktörü içermeyen hiPSCs türetmek için bir protokol mevcut. Şekil 1A MEF&#39;ler bir tabakası üzerinde STEMCCA yaklaşımı ile yeniden programlama sonra, üç farklı önceden kesilmiş hiPSC hatları temsili görüntüsünü gösterir. STEMCCA yeniden programlama yaklaşımının temel avantajı, farklı araştırma grupları ve yerlerde, birden fazla bilim adamları tarafından elde tutarlı yeniden progr...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Türetilmesi ve Karakterizasyonu bir Transgen ücretsiz İnsan Kaynaklı Pluripotent Tanımlı Klinik dereceli Koşullar içine Hücre Hattı ve Dönüşüm Kök

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K Görüntüleme.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

Video: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biyoloji

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 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 ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Sitometrisi ile Yüksek Verim kardiyomiyositlerde İnsan pluripotent kök hücrelerin Farklılaşma ve Karakterizasyonu

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

İnsan Antral Foliküllerinden Soya Özgü Hücrelerin Ekstraksiyonu, Etiketlenmesi ve Saflaştırılması

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...