Bu yazıda anlatılan çalışma UCR Kök Hücre Çekirdek için Rejeneratif Tıp California Institute (CIRM) fon ile mümkün olmuştur.

Tüm protokol GeneJuice (EMD) ve bir AMAXA nuclefector cihazı kullanılarak insan iPSCs arasında nucleofection kullanılarak insan iPSCs transfekte etmek için protokoller takip besleyici içermeyen kültür, insan iPSCs adapte etmek için bir yöntem ile başlar. Not: Aşağıdaki prosedürler steril bir laminer akış kaputu yapılmaktadır. Tüm ortamlar ve çözümler 37 ° C&#39;de veya başka bir şekilde belirtilmediği sürece, başlamadan önce oda sıcaklığına gelmesi vardır. 1. Besleyici-free sistem insan iPSCs Kurulması Önceden besleyici hücreler üzerinde muhafaza İnsan iPSCs Geltrex kaplı çanak aktarılır ve önceki besleyici ücretsiz transfeksiyon iki pasajlar için muhafaza, bölünebilir. <ol><li> 4 ° C&#39;de gece boyunca Geltrex çözülme Geltrex kaplama hazırlamak için, soğuk DMEM çözülmüş Geltrex 01:50 sulandırmak. Yavaşça çözümleri karıştırın. </li></ol> Not: Geltrex, Matrigel gibi bazal membran MATR bir çözünür biçimidirmurin gelen ix saflaştırılmış Engelbreth-Holm-Swarm (EHS) tümör hücreleri. Alternatif olarak, Matrigel besleyici içermeyen insan IPSC kültürlerinin kurulması için bir hücre dışı matris olarak kullanılabilir. <ol start="2"><li> Geltrex çözüm (35 mm de için 1 ml) ile kültür kuyuların tüm yüzeyi kaplayın. 37 Geltrex ile kaplayın kuyuları ° C de 1 saat inkübatör. </li><li> Geçit insan iPSCs için, başına iyi accutase 1 ml ekleyin ve çoğu hücreleri ayırmak başlayana dek ° C 1 dakika 37 inkübe. </li></ol> Geçit insan iPSCs için, başına iyi accutase 1 ml ekleyin ve çoğu hücreleri ayırmak başlayana dek ° C 1 dakika 37 inkübe. <ol start="4"><li> Hücreleri ve yavaşça girdap plakası 10-15 cam boncuk ekleyin. KnockOut DMEM / F12 2 ml ekleyin ve hafifçe çiğnemek. 10 ml lik santrifüj tüpüne hücre süspansiyonu aktarın. </li><li> Oda sıcaklığında 3 dakika için 800 rpm&#39;de hücreleri Spin. </li><li> İnsan IPSC pelet zedelemeden, tüpten süpernatant aspire. Yavaşça tüp fiskehücre pelletini dağıttı. </li><li> Kaplamalı kuyudan Geltrex çıkarın. </li><li> Yavaşça STEMPRO uygun bir hacim içinde insan IPSC pelet tekrar süspansiyon. ) Proliferasyon hızına bağlı olarak, besleyiciler kuyuları arasındaki dağıtın. İnsan iPSCs 1:02-1:06 arasında bölünmüş bir oran içinde pasajlanmağa devam edilebilir. </li><li> Dikkatle yerde% 5 CO 2 inkübatör, dikkatle girdap plakası kuyuların genelinde hücreler eşit bir şekilde dağılımını sağlamak için içine. </li><li> Hücreler kadar günlük Yem hücreleri tekrar Bölünecek hazır (hücrelerin% 80 confluency ulaştığınızda). </li><li> 1:2 bölünmüş oranı yeni Geltrex kaplı sıra üzerine Passage insan iPSCs (adım 1,3-1,9). Küçük koloniler kurdu ve iyi Geltrex kaplı transfeksiyon öncesinde üzerinde eşit dağıtılmalıdır. </li></ol> 2. GeneJuice ile insan iPSCs transfeksiyonu Hücreler (6 oyuklu plakalar üzerinde yetiştirilen) optimum transfeksiyon verimliliği elde etmek için transfeksiyon gününde konfluent yaklaşık olarak% 40 -50 olmalıdır. It isdeğil ertesi güne kadar hücre orta değiştirmek için gereklidir. <ol><li> Steril bir 1,5 ml Eppendorf tüp 100 ul KO-DMEM/F12 hazırlayın. 27 ul GeneJuice transfeksiyon reaktif ekleyin. İyice karıştırın. 5 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 4 ug plazmid DNA ekleyin. 15 dakika süre ile oda sıcaklığında inkübe tüp. Plazmid tercih optimum transfeksiyon etkinliği için kritik önem taşır. Bu promotör 5 (pCAG-eGFP) CAG tarafından tahrik edilen bir geliştirilmiş yeşil flüoresan protein (eGFP) sahip bir plazmid kullanır. Promotörü CAG insan iPSCs içinde transkripsiyonel olarak etkin ve bu nedenle, bu hücrelerde transgen ekspresyonu sürmek için kullanılan güçlü bir kurucudur. Bizim ellerde, plazmid doğrusallaştırma transfeksiyon verimliliği etkileyecek görünmüyordu. </li><li> Hücreler ve girdap plaka GeneJuice-DNA karışımı ekleyin. Kuyu insan iPSCs ile transfeksiyon karışımı temasını artırmak için 5 dakika için 1200 rpm (&#39;spinoculation&#39;) yöntemini de plaka Spin. </li><li> 37 &amp; adresindeki hücreleri inkübedeg; C gecede. </li><li> Bir sonraki gün transfeksiyon etkinliği için izler. </li><li> Kararlı transfeksiyon için, taze STEMPRO ile ertesi gün ortam değişir. 48 saat - 24 saat sonra uygun antibiyotik seçimi ekleyin. </li></ol> 3. Insan iPSCs arasında Nucleofection Besleyiciler veya Geltrex üzerinde yetiştirilen insan iPSCs nucleofection için de kullanılabilir. Ancak, güçlü fare embriyonik fibroblast (MEF) ya da insan sünnet fibroblast (HFF) yüksek hücre canlılığı ve iyileşme sağlamak için besleyiciler üzerine nucleofected insan iPSCs replating öneririz. En az 2 milyon hücre nucleofection takiben yüksek hücre canlılığı elde etmek için kullanılmalıdır. <ol><li> Gecede besleyici hücreler ile Serum Değiştirme (KOSR) medya, insan IPSC KnockOut bekletilmeleri ile besleyici hücre klimalı ortam (CM) hazırlayın. CM 24 saatte toplayın. </li></ol> Not: besleyici tabakalar (örneğin, BLK6, CF1 ve MF1 gibi) hazırlanması için kullanılan herhangi bir fare suşu fo kullanılmaya uygundurr CM yapma. <ol start="2"><li> Nucleofection gününde, en az 1 saat için 10 uM ROCK inhibitörü olan insan iPSCs ön-tedavi. </li><li> Steril bir 1,5 ml Eppendorf tüp, insan kök hücre nucleofector çözümü 82 ul hazırlayın. 18 ul takviyesi 1 ekleyin. İyice karıştırın. 5 dakika için 37 ° C sıcaklıkta çözelti inkübe edin. </li><li> Pre-sıcak besleyici hücre klimalı ortam (CM) ve% 0.25 tripsin / EDTA 37 ° C&#39;ye kadar Kaputun altında, prelabel tek steril 15 ml konik tüp. </li><li> Dikkatle nucleofected edilmesi için insani IPSC kültür ortamı çıkarın. Yavaşça kuyu başına 2 ml 1X fosfat tamponlu çözelti (PBS) ile yıkayın hücrelerini. PBS aspire. Kuyu başına% 0.25 tripsin 1 ml ekleyin. 3 dakika için 37 ° C &#39;de hücreler inkübe edin. </li><li> Yavaşça 1000 ml pipet ile hücreleri çiğnemek. Tüm insan iPSCs tamamen müstakil emin olmak için kuyunun dibinde yıkayın. Etiketli 15 ml konik tüp hücre süspansiyonu aktarın. </li></ol> Not: tek c içine trypsinizationarşın kesinlikle kaçınılmalıdır. Hücrelerin yaklaşık olarak tripletler oluşan Hücreler tek hücre küçük öbekler halinde uzaklaştırılabilir olması gerekmektedir. Hücrelerin küçük öbekler (hücre üçe) hücrelerin daha sonraki kullanım sırasında tek bir hücre içine ayrılmış olacaktır. <ol start="7"><li> Tripsin inaktive 9 ml MEF ortam ekleyin. 3 dakika için 800 rpm&#39;de hücreleri Spin. Dikkatlice süpernatant aspire ve hücre pelet gibi bırakabilirsiniz. </li><li> Adım 3.3 insan kök hücre nucleofector çözümü önceden ısıtılmış 100 ul hücreleri yeniden süspanse edin. </li><li> Bir 1 ml pipet kullanarak nucleofector küvetine hücreleri aktarın. </li><li> Küvet içinde hücre süspansiyonu içine plazmid DNA 4 ug ekleyin. Nazik bir döndürme hücreleri ve DNA karıştırın. Kaputun yüzey üzerinde iki küvet dokunun. </li><li> Nucleofector tutucu içine küvetini takın. Programları B-016 kullanın. Düğmesine X. basarak Nucleofect hücreleri </li></ol> &lt; <ok> Nucleofection işlem komple olduğunda&gt; gösterilired (genellikle 1-5 saniye sürer). Diğer nucleofection programları kullanımı (A-023, A-033 ve U-023) insan iPSCs dan eGFP-pozitif hücrelerinin% 10&#39;undan daha az bulunmuştur. </ok><ol start="12"><li> Sağlanan Pasteur plastik pipet kullanarak küvet gelen nucleofected hücreler al. Steril bir 1,5 ml Eppendorf tüp içine önceden ısıtılmış CM ve ROCK inhibitörü bunları resuspending hücreleri kurtarın. Hücreler kurtarmak izin vermek için 10 dakika için 37 ° C &#39;de hücreler inkübe edin. </li><li> 1 ml pipet kullanarak besleyici tabakalar üzerine açılan bilge Transferi hücreler. 37 ° C&#39;da gece boyunca hücreler inkübe edin. </li><li> Bir sonraki gün transfeksiyon etkinliği için izler. Transgenik insan iPSCs istikrarlı klonları türetmek için stabil CM ve ROCK inhibitörleri ile ertesi gün orta değiştirmek, transgen ifade. 48 saat - 24 saat sonra uygun antibiyotik seçimi ekleyin. </li></ol> 4. Temsilcisi Sonuçlar: <img alt="Şekil 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <strong&gt; Şekil 1. pCAG-eGFP ile transfekte Riv1 insan iPSCs arasında fotomikrograflarıdır. (A) eGFP-Salgılayan hücrelerin Riv1 Geltrex 12 saat sonrası transfeksiyon üzerinde GeneJuice kullanılarak transfekte. Riv1 insan iPSCs kolonileri kaplama ve B-016 (B) ve A-023 (C) programını kullanarak nucleofection takip besleyiciler üzerinde oluşmuş. (D) her yerde bulunan bir eGFP ifade ile stabil eGFP ifade eden insan IPSC kolonileri GeneJuice türetilmiştir. (E) stably transfekte Riv1 insan iPSCs embriyoid vücudu farklılaşması esnasında bünye eGFP ifade korudu.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

Çıkar çatışması ilan etti.

Basit, sağlam ve son derece tekrarlanabilir teknikleri Bizim protokoller sonucunda belirgin toksik etkisi ve hücre ölümüne olmadan insan iPSCs transgenlerin tanıtmak. İnsan iPSCs hücrelerin daha küçük kümeleri (5-10 hücreleri) içine pasajlandı ve çok sayıda küçük koloniler optimum transfeksiyon etkinliği sağlamak için yüksek yoğunluklu (1:2) de Geltrex üzerinde kaplanacaktır. Farklılaşma ve hücre ölümü daha yatkın insan IPSC hatları için, insan iPSCs yüksek sayıda (en fazla 4 X 10 6 hücre) tek bir nucleofection deney için kullanılmalıdır. Geçici transfeksiyon deneyinde 1 gün içinde transgen ifade eden insan iPSCs çok sayıda üretir. Stably transfekte IPSC klonlar genellikle 7 gün içinde ortaya çıkar ve bu transgenik koloniler üç hafta içinde iade için hazır olmalıdır. CAG organizatörü kullanımı eGFP muhabirin her yerde ifade edilmesini sağlayacaktır burada açıklanan. Bu tür gelişmeler altında, bizim protokolleri ekspresyonu, c dahil olmak üzere, diğer uygulamaların içine beslenebilironditional indüksiyon, soy özgü muhabiri hatları, shRNA veya siRNA knockdown türetilmesi, gen hedefleme ve homolog rekombinasyon.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> EDTA ile% 0.25 tripsin </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2-merkaptoetanol </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3 mm cam boncuklar </td><td> Fisher Scientific </td><td> 11-312A </td><td> Cam boncuklar, sodyum hidroksit (NaOH) ve saf su ile durulanır gece boyunca hidroklorik asit (HCl) ile yıkandı, ve kullanımdan önce otoklav ile sterilize edilmesi gerekir. </td></tr><tr><td> Accutase hücre ayrışma reaktifi </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> Temel fibroblast büyüme faktörü (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (yüksek glukoz) </td&gt; <td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> Fetüs buzağı serumu </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> Büyüme faktörü azalır </td></tr><tr><td> GeneJuice transfeksiyon reaktif </td><td> EMD Biosciences </td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I (100X) </td><td> Invitrogen </td><td> 35050061 </td><td> L-Glutamin (Invitrogen, 25030081) de yerine kullanılabilir. </td></tr><tr><td> İnsan IPSC KOSR medya </td><td></td><td></td><td> 500 ml insan ES ortam 390 ml KnockOut DMEM / F12, 100 ml KnockOut serum Replacer, 5ml Glutamax I (100X), 5ml NEAA (100X), 500 ul 2-merkaptoetanol (55 mm) oluşan ve 10 ng / ml bFGF ile desteklenmiş . </td></tr><tr><td> KnockOut DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td>KnockOut Serum Değiştirme (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Fare embriyonik fibroblast medya </td><td></td><td></td><td> EMB ortam% 90 FBS, 1X NEAA, 1X Glutamax I ve DMEM içinde 1X sodyum piruvat (yüksek glikoz) oluşmaktadır </td></tr><tr><td> Sigara esansiyel amino asit, NEAA? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> İnsan kök hücre nucleofector solüsyonu 1 takviyesi ile </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> Fosfat Ca 2 olmadan + ve Mg 2 Tamponlu Salin + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> Sodyum piruvat (100X) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO orta kiti </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ml STEMPRO ortam 454 ml Knock oluşmaktadırÇıkış DMEM / F12, 10 ml serum içermeyen STEMPRO büyüme ek (50X), 5ml Glutamax I (100X), 5ml NEAA (100X), 36 ml BSA (bovin serum albumin, 25%), 909 ul 2-merkaptoetanol (55mm ) ve 10 ng / ml bFGF ile desteklenmiş. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

Genetik modifikasyon kök hücre biyolojisi okuyan ve insan embriyonik kök hücreleri (hESC) 1 potansiyel klinik uygulamaları ortaya koyan önemli bir araç olmaya devam etmektedir. Birkaç gen teslimat yöntemleri geliştirmeleri 2-9 tarif edilmiş olmasına rağmen, transfeksiyon hESC için kaprisli bir süreç olmaya devam etmektedir ve henüz insan kaynaklı pluripotent kök hücreler (iPSCs) rapor edilmemiştir. Bu videoda, bizim laboratuarımızda rutin transfects ve gelişmiş yeşil flüoresan protein (eGFP) muhabiri ile plazmid kullanılarak insan iPSCs nucleofects nasıl gösterilmektedir. İnsan iPSCs uyarlanmış ve besleyici hücre transfeksiyon olasılığını ortadan kaldırmak için ve transfeksiyon sonrasında stabil transgenik IPSC klonların verimli seçimine izin veren besleyici ücretsiz kültürler olarak korunur. Nucleofection, insan iPSCs besleyici hücre Klimalı besleyiciler üzerinde nucleofected ve replated hücrelerin küçük öbekler içine tripsinize ROCK inhibitörü 11 ile ön tedavi edilirhücre kurtarma geliştirmek için orta. Transgen ifade eden insan iPSCs 6 saat sonra elde edilebilir. Antibiyotik seçimi 24 saat stabil transgenik hatları 1 hafta içinde görünür sonra uygulanır. Tüm protokol, sağlam ve bu hücrelerin pluripotency değiştirmeden, insan IPSC hatları için röprodüksiyon özelliğine sahiptir.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetik modifikasyon alanında son gelişmelere rağmen, insan embriyonik kök hücreleri (hESC) aktarılmasının kaprisli bir süreç olmaya devam etmektedir. Bildiğimiz kadarıyla, insan kaynaklı pluripotent kök hücreler (iPSCs) transfekte etmek için sistematik ve etkili bir yöntem bildirilmemiştir. Burada, biz verimli transfekte ve insan iPSCs nucleofect sağlam protokoller açıklanmaktadır.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

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JoVE Journal

Biology

21.4K Görüntüleme.  University of California Riverside. Genetik modifikasyon alanında son gelişmelere rağmen, insan embriyonik kök hücreleri (hESC) aktarılmasının kaprisli bir süreç olmaya devam etmektedir. Bildiğimiz kadarıyla, insan kaynaklı pluripotent kök hücreler (iPSCs) transfekte etmek için sistematik ve etkili bir yöntem bildirilmemiştir. Burada, biz verimli transfekte ve insan iPSCs nucleofect sağlam protokoller açıklanmaktadır.

Video: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofector İnsan Sinir Kök Hücreler Transfecting

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Biyoloji

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Kültürleme İnsan Embriyonik ve Bağlı Pluripotent Kök Hücrelerinin için uygundur Fare embriyonik fibroblast hücreleri hazırlanması

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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, ...

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 ...