Biz el yazması değerli tartışmalar için Lee laboratuar üyelerine teşekkür etmek istiyorum. Lee laboratuarda çalışma Hücre Araştırma Fonu (Tedco) Kök New York Kök Hücre Vakfı Robertson Araştırmacı Ödülü ve Maryland hibe tarafından desteklenmiştir.

1.. Hücre hazırlanması ve Ortam (Gün 1) <ol><li> Kültür ve DMEM ortamı,% 10 FBS ihtiva eden insan fibroblast genişletmek. </li><li> Plaka insanlara özgü fibroblastlar; transdüksiyon bir gün önce oyuk başına uygun bir yoğunlukta bir 24-yuvalı plaka üzerine (Şekil 1). NOT: farklı hücre tipleri farklı bağlanma yeteneği var çünkü şu seri seyreltme (200K, 100K, 50K, 25K, 12.5K ve 6.25K) önerilir. </li><li> Hücreler tamamen yapışmış ve genişletmiştir sağlanması, bir 37 ° C,% 5 CO2 inkübatörü içinde bir gün için hücreler inkübe edin. </li></ol> 2.. İletimi yapın (2. Gün) <ol><li> Transdüksiyon gününde, hücre yoğunluğunu kontrol etmek ve en etkin yoğunluğu kuyu (Şekil 2) seçtik. Yüksek (80 ~% 90), orta (50 ~% 70) ve düşük (20 ~ 40%): Bu üç farklı yoğunlukları almak daha iyidir. </li><li> En az 1 saat önce transdüksiyon, fibrobl aspireast hücrelerden ortam ve fibroblast ortamın yeni bir 300 ul değiştirin. </li><li> -80 ° C depolama 4 farklı Sendai virüs tüplerin bir set çıkarın. Birkaç saniye için bir 37 ° C su banyosu içinde, aynı zamanda, her tüp çözülme ve sonra su banyosundan tüpleri alır. Daha sonra, oda sıcaklığına kadar bunları çözülme; santrifüj 10 saniye boyunca 6.000 xg&#39;de tüpler ve kullanıma kadar buz koyun. -Donma yeniden ve titreleri korumak değil çünkü virüs cozundurmeden. </li><li> Mikro santrifüj tüpüne dört Sendai virüsü boruların (Ekim-4, Klf-4, c-Myc ve Sox-2) her birinin, belirtilen miktarlar ekleyin. Çözelti hafifçe pipetleme iyice karıştırılır olduğundan emin olun. Örneğin, transdüksiyon için 24 oyuklu plaka içinde LOOKWELL 50K hücre / bir de: (Life Technologies Analiz Sertifikalarını dayalı Titer, partiden diğer partiye farklı olabilir) Tüp hOct-4 = 6.0 x 10 7 UKÜ, 3 İçişleri Bakanlığı 5 ul = Tüp hSox-2 = 6.5 x 10 7 CIU, 3 MOI = 4.6 μ; L Tüp hKlf-4 = 6.3 x 10 7 UKÜ, 3 İB = 4.8 ul Tüp hc-Myc&#39;nin = 7.8 x 10 7 UKÜ, 3 İB = 3.8 ul Toplam = 24 oyuklu plakanın dört virüs faktör karışımı / bir de (50K hücreleri) 18.2 ul </li><li> Hücre yoğunluklarına göre; 2X, 1X ve kuyunun içine virüs karışımı 0.5x hacim eklemek. Yavaşça, plaka önden arkaya sallayın sol ve sağ (2X: virüs karışımı, 1X 36.4 ul: 18.2 ul ve 0.5x: 9.1 ul). </li><li> 37 ° C,% 5 CO2 inkübatöründe hücreleri koyun ve bir gece boyunca inkübe edilir. </li></ol> 3.. Kültür Orta Değiştirme (Gün 3 &amp; Day 5) <ol><li> 24 saat transdüksiyon sonra, 500 ul taze fibroblast ortam ile orta yerine. </li><li> 5. günde, taze fibroblast medya ile Orta değiştirin. </li></ol> 4.. Co-kültür için MEF Yemekleri hazırlayın (Gün 8) <ol><li> % 10 ile 60 mm kültür tabaklarında MEF hücreleri hazırla FBS DMEM ortamı bir da ihtivaMEF besleyici-hücreleri üzerine transdüse fibroblast pasaj önce y. Not: Her 60 mm çanak MEF 5 x 10 5 kullanıyorsunuz. </li></ol> 5.. Başlangıç ​​Co-kültür MEF besleyici hücreler (Gün 9) ile Hücreleri kalıt <ol><li> Transduced hücreleri kaplama önce, aspirat% 10 FBS MEF besleyici yemekler DMEM ortamı bulunan ve FBS DMEM ortamı ihtiva eden taze% 10 ekleyin. </li><li> 24 oyuklu bir plaka içerisinde olan ve D-PBS ile bir kez yıkama hücreleri transduse fibroblastlardan orta çıkarın. 24-çukurlu plaka inthe fibroblast hücreler içine% 0.25 tripsin / EDTA, 200 ul ekleyin. Hücreler plakadan kopmaya başladığından zaman tripsin / EDTA, birlikte en az 5 dakika inkübasyondan sonra, 15 ml konik bir tüp içinde büyüme ortamı ile hücreleri toplamak. Daha sonra 4 dakika boyunca 6,000 x g de santrifüj. NOT: 2X, 1X ve 0.5X virüs Havuzlanması transdük fibroblast tavsiye edilir. </li><li> Süpernatan Ortamı çıkarın ve yeniden askıya yıkamatripsin ile nötrleştirme için taze ortam fibroblast 4-5 ml hücre topağı. Daha sonra 4 dakika boyunca 135 x g&#39;de santrifüj. </li><li> Örneğin 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 ve 1/128 60 sayısı: seri seyreltme yoğunluğu olarak ve MEF üzerine fibroblast ortam ile birlikte kültür için başlangıç ​​Plaka hücreleri mm çanak. Transdüksiyon ilk haftasında hücre ölüm oranı ile ilgili olarak, bir 1/2 veya 1/128 yoğunluğa seyreltilmesi gerekmeyebilir. Transgen RT-PCR için bir pozitif kontrol olarak, toplam RNA çıkarmak için bir kenara kalan hücrelerin koyun. </li><li> % 5 CO2 inkübatör gece boyunca, 37 ° C&#39;de 60 mm yemekler yerleştirin. </li></ol> 6.. İnsan Embriyonik Kök Hücre Orta Yem ve Hücreleri (Gün 10) Monitör <ol><li> Ertesi gün, 10 uM Y-27632, insan ES ortama fibroblast ortamı değiştirmek. Bundan sonra, insan ES ortamı ile günlük orta değiştirin: 800 ml DMEM/F12 + 200 mi Knockout Serum Replacement + 10 ml L-glutamin, 10 mM + 10 minimum MEM esansiyel olmayan amino asitler miTemel Fibroblast Büyüme Faktörü 2-mersaptoetanol + 10 ng / ml + 1 ml. </li><li> Taze insan ES medya ile bir günlük medya değişiklik yapmak. Bir hafta sonra, koloni ES-benzeri hücre kümeleri oluşumu için bir mikroskop altında bir kez toplam 2 ~ 3 gün bulaşıkları kontrol edin. Koloniler bulundu önce hücre tipine bağlı olarak, kültür daha çok veya daha az zaman alacaktır. </li></ol> 7. Uyarılmış pluripotent hücre kolonileri Kök ve Hücreleri genişletin Toplama (Gün 20 ~) <ol><li> Üç hafta sonra, transdüksiyon, koloni toplama için uygun yetiştirilen görünmelidir. </li><li> Koloni toplama bir gün önce, 24 oyuklu bir plaka içerisinde MEF besleyici hazırlar. NOT: Biz bir 24-plaka içinde MEF 12.5 x 10 5 kullanıyorsanız. </li><li> Ertesi gün, 10 uM Y-27632, insan ES ortama fibroblast ortamından MEF yemekler orta değiştirin. Elle toplama başlığı içinde mikroskop altında her zaman bir koloni almak ve pipetleme küçük kümeleri yapmak ve bunları aktarmakyeni birçok klon almak için MEF dishes.Try hazırlanan üzerine. </li><li> Geçidi ve başlangıçta bir 6 yuvalı plakaya bir 24-çukurlu plaka her bir genişletme. Daha sonra, 6-çukurlu plaka yayılımıyla önce, 60 mm tabaklar için. </li></ol> (10 geçiş sonrası) İnsan iPSCs 8. Karakterizasyonu <ol><li> İmmünofloresan <ol><li> Günde ortam değişikliği ile 4-5 gün boyunca 24 oyuklu plaka ve kültür Plate insan iPSCs. </li><li> Imüno-flüoresan deneyi çalıştırmak için, bir kere D-PBS ile plakayı yıkayın. Daha sonra 30 dakika boyunca% 0.4 paraformaldehid içinde hemen hücreleri gidermek. Daha sonra 5 dakika süre ile D-PBS içinde üç kez yıkayın. </li><li> Oda sıcaklığında 15 dakika boyunca PBS içinde% 0.1 Triton X-100 çözeltisi ile işlem görmüş hücrelerin tedavi. </li><li> Permeablization çözeltisi (% 0.1 triton X-100 çözeltisi) aspire, daha sonra oda sıcaklığında 1 saat boyunca% 0.5 BSA bloke çözeltisi ilave edilir. </li><li> Nanog&#39;un, Ekim-4, SSEA (evre özgü embriyonik ANTIG bir algılama gerçekleştirinen) ve TRA-1-81,% 0.5 BSA çözeltisi içinde bir anti-insan antikoru kullanılarak (tümör rejeksiyon antijen) (antikor seyreltme oranı için malzeme tablosunu kontrol edin). Oda sıcaklığında 2 saat için primer antikor inkübe edin. Daha sonra 5 dakika her biri için D-PBS içinde üç kez yıkayın. </li><li> Oda sıcaklığında% 0.5 BSA çözeltisi içinde 1:2,000 seyreltilmiştir 2. antikor olarak Alexa Fluor 488 keçi anti-fare IgG antikorları ile inkübasyondan 1 saat sonra floresan mikroskobu kullanılarak kontrol edin. DAPI ile her bir besleyici hücreleri ve MEF hiPSCs tüm çekirdek Leke. </li></ol></li><li> Transgen Teyit RT-PCR <ol><li> RNA ekstraksiyon reaktifleri kullanılarak hücre pelletinden toplam mRNA ekstrakte edin. </li><li> Ters transkriptaz ile PCR amplifikasyonu için toplam RNA ve elde edilen cDNA alikotları üzerinde bir ters transkripsiyon (1 ug) yapın. </li><li> CDNA şablon 2 ul, 2x PCR Master Mix 10 ul, primerler 2 pmol 2 ul içeren PCR karışımları hazırlamak(Ileri: 1 ul ve ters: 1 ul) ve DW 6 ul. Tablo 1 &#39;de sıralanan primerler ile RT-PCR yapmak, gen ekspresyonunu tespit etmek için. </li><li> Amplifikasyon reaksiyonları verimliliği için bir kontrol olarak, GAPDH geni ile RT-PCR&#39;ler gerçekleştirin. Görselleştirme ve% 1 agaroz jel elektroforezi ile analiz PCR ürünü. </li></ol></li></ol>

<ol>
	<li>Yu, J., 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>Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mouse+induced+pluripotent+stem+cells+without+viral+vectors.">Generation of mouse induced pluripotent stem cells without viral vectors.</a> Science. 322, 949-953 (2008).</li><li>Zhou, W., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27, 2667-2674 (2009).</li><li>Woltjen, 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=piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells.">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458, 766-770 (2009).</li><li>Okita, 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8, 409-412 (2011).</li><li>Yusa, K., Rad, R., Takeda, J., Bradley, A. <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+induced+pluripotent+mouse+stem+cells+by+the+piggyBac+transposon.">Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.</a> Nature Methods. 6, 363-369 (2009).</li><li>Kim, 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=Generation+of+human+induced+pluripotent+stem+cells+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Yu, J., 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=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Yoshioka, N., 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+Generation+of+Human+iPSCs+by+a+Synthetic+Self-Replicative+RNA.">Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA.</a> Cell Stem Cell. 13, 246-254 (2013).</li><li>Dowey, S. N., Huang, X., Chou, B. K., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+integration-free+human+induced+pluripotent+stem+cells+from+postnatal+blood+mononuclear+cells+by+plasmid+vector+expression.">Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.</a> Nature Protocols. 7, 2013-2021 (2012).</li><li>Fluri, D. 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=Derivation,+expansion+and+differentiation+of+induced+pluripotent+stem+cells+in+continuous+suspension+cultures.">Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures.</a> Nature Methods. 9, 509-516 (2012).</li><li>Nakagawa, 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+without+Myc+from+mouse+and+human+fibroblasts.">Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.</a> Nature Biotechnology. 26, 101-106 (2008).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> P Jpn Acad B-Phys. 85, 348-362 .</li><li>Silva, S. S., Rowntree, R. K., Mekhoubad, S., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome+inactivation+and+epigenetic+fluidity+in+human+embryonic+stem+cells.">X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells.</a> P Natl Acad Sci USA. 105, 4820-4825 (2008).</li><li>Kim, 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=miR-371-3+expression+predicts+neural+differentiation+propensity+in+human+pluripotent+stem+cells.">miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.</a> Cell Stem Cell. 8, 695-706 (2011).</li></ol>

Yazarlar ifşa hiçbir şey yok.

HiPSCs insan somatik hücreleri yeniden programlama temel biyoloji, kişisel tıp ve transplantasyon 12 görülmemiş sözleri tutar. Daha önce, insan iPSC nesiller gibi ilaç geliştirme ve nakli tedavileri 13 gibi diğer klinik uygulamaları sınırlandıran istenmeyen genetik mutasyonlar oluşturabilir konakçı genomu içine entegrasyon riski DNA virüsü gereklidir. Bu nedenle birlikte, birçok çalışmada çeşitli alternatif yöntemlerle vektör ve transgen-free sistemi, insan iPSCs oluşt...

İnsan embriyonik kök hücreleri (hESC) ilaç taraması için, hastalık modelleme için potansiyel olarak yararlı olabilir ve hastalık ve doku yaralanmaları tedavi etmek için hücre tabanlı tedaviler geliştirmek için kendini yenilemek in vitro ve sahip pluripotensin bir kapasiteye sahip. Ancak, HESC çünkü, immünolojik onkolojik ve etik engellerin hücre replasman tedavisi için bir sınırlama var, ve hastalık ile ilgili genlerin çalışma, hastalığa özgü HESC pre-implantasyon genetik tanı (PGD) yaklaşımları ile izole edilebilir, ama yine de teknik açıdan zor ve embriyo bağışlar oldukça nadirdir. Bu sorunlar, uyarılmış pluripotent kök hücreler (hiPSCs) gelişmesine yol açmıştır kök hücre biyolojisi, ilerleme ile ilgilidir. İnsan iPSCs genetik yetişkin insan somatik hücrelerinden yeniden programlanan ve onları böyle d gibi rejeneratif tıp için yararlı bir kaynak yapar HESC benzer pluripotent kök hücre benzeri özellikler, liman vardırhalı keşif, hastalık modelleme ve hastaya özgü şekilde 1,2 hücre tedavisi. Bugüne kadar, virüs aracılı (retrovirüs ve adenovirüs) dahil olmak üzere insan iPSCs oluşturmak için çeşitli yöntemler, 3, non-virüs aracılı (BAC sistemi ve vektörler transfeksiyon) 4 gen transdüksiyon ve protein dağıtım sistemi 5-7 vardır. Virüs-aracılı gen iletim verimliliği belirli bir düzeyde olmak, ancak bunlar, kontrolsüz bir şekilde yeniden programlama genleri ifade etmek için bir ev sahibi kromozomları içine entegre, çünkü viral vektörler, genetik iz bırakabilir. Transkripsiyon faktörlerinin viral entegrasyonu etkinleştirmek veya konak genleri 8 inaktive olabilir bile, bu beklenmedik bir genetik sapma ve tümörgenez 5,9 riskini neden olabilir. Öte yandan, somatik hücrelere protein veya RNA doğrudan katılması bildirilmiştir, ancak bu emek-yoğun, tekrarlı transf gibi bazı dezavantajları vardır edildiection ve 7,10 yeniden programlama düşük seviyesi. Hatta epizomal olmayan entegre adenovirüs, adeno-bağlantılı virüs, ve plasmid vektörler yine 11 göreli olarak daha az verimlidir. Bu nedenlerden dolayı, iPSC nesil yüksek etkinlik ve daha az genetik anormallikler olmayan entegrasyon yeniden programlama yöntemlerini seçmek için makul. Bu çalışmada, bir-Sendai virüs esasına dayalı yeniden programlama kullanın. Bu yöntem, konakçı genomuna entegre olmayan ve tutarlı bir şekilde, transgen entegrasyon olmadan insan iPSCs üreten olduğu bilinmektedir.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

Birkaç yıl önce, insan uyarılmış pluripotent kök hücreler (iPSCs) kurulması biyomedikal yeni bir dönem başlatmıştır. Insan iPSCs potansiyel kullanımları, insan genetik hastalıklarının modellenmesi patogenezi, gen düzeltme sonrası otolog hücre tedavisi ve hastaya özgü ve belirti gerekli hücrelerin bir kaynağı sağlayarak kişiselleştirilmiş ilaç tarama içerir. Ancak, bu tür bir insan iPSCs üretim sonrası kalan yeniden programlama faktörü transgen ekspresyonunu ortadan kaldırarak üstesinden gelmek için çok engel vardır. Daha da önemlisi, farklılaşmamış insan iPSCs kalıntı transgen ifadesi doğru farklılaşmaları engel ve hastalıktan ilgili vitro fenotiplerinde yorumunu şaşırtıp-saptırırlar olabilir. Bu neden ile, entegrasyon içermeyen ve / veya transgen içermeyen insan iPSCs adenovirüs, piggyBac sistemi minicircle vektör epizomal vektörler, direkt protein temini gibi çeşitli yöntemler kullanılarak geliştirilmiş ve mRNA sentezlenmiştir. Bununla birlikte, reprogra etkinliğientegrasyon-ücretsiz yöntemlerle features çoğu durumda oldukça düşüktür. Burada, Sendai-virüsü (RNA virüsü) tabanlı yeniden programlama sistemi kullanarak insan iPSCs izole etmek için bir yöntem mevcut. Bu yöntem, yeniden programlama tutarlı sonuçlar ve düşük maliyetli bir şekilde yüksek bir verimlilik göstermektedir.

Genellikle enfekte fibroblastlar Sendai virüs iletiminde sonra birkaç gün içinde herhangi bir morfolojik değişiklik görünmüyor, ancak beş gün sonra, farklı şekiller (Şekil 1) sahip başlar. Şekil 1, bir sağ panelde tarif edildiği gibi, hücreler, daha fazla tipik fibroblast morfolojiye sahip değildir. Bunlar sitoplazmada daha yuvarlak bir şekil ve büyük çekirdeği vardır. Transdüksiyon% 80 hücresel confluency gerçekleştirildiğinde bile onlar yeniden programla...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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

Burada, biz tutarlı sonuç ve geliştirilmiş verimlilik gösterir Sendai virüs ile transgen-özgür insan iPSCs içine insan somatik hücreleri yeniden programlamak için kurulan yöntem sunuyoruz.

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Biology

22.2K Görüntüleme.  Johns Hopkins University School of Medicine.  Burada, biz tutarlı sonuç ve geliştirilmiş verimlilik gösterir Sendai virüs ile transgen-özgür insan iPSCs içine insan somatik hücreleri yeniden programlamak için kurulan yöntem sunuyoruz. 

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

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

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

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

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

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

Biyoloji

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 Mice Derived from Induced Pluripotent Stem Cells

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

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

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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

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

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

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

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

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

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.