Not: steril kültür koşullarını korumak için, bu protokol tüm prosedürlerin steril laboratuar uygulamaları kullanılarak gerçekleştirilen ve laminer akış kaputu altında yürütülmelidir. Başlamadan önce, herhangi bir ortam, 37 dengelenmiş olduğundan emin ° C ve uygun gazladı. Geltrex kaplı Kültür Yemekleri hazırlanması Not: CELLstart kaplı kültür kaplarına kullanımı için Ek <ol><li> Çözülme Geltrex yavaş yavaş 2-8 (1 mL) ° C tüp ve Knockout D-MEM/F-12 99 ml 1:100 onu sulandırmak. Çözüm hafifçe karıştırın. Not: Bazı iPS hücre hatları, optimum büyüme için farklı bir Geltrex seyreltme gerekebilir. Alternatif dilüsyonları için eke bakınız. </li><li> Her kültürün çanak tüm yüzeyi Geltrex çözüm (35 mm yemek için 1 ml, 60 mm yemek için 1.5 ml) ile kaplayın. </li><li> Kurumasını önlemek ve 37 yemekleri az 1 saat inkübe Parafilm ° C ile her tabak sızıntısını önleyiniz Not: Bu noktada, 1 aya kadar 4 ° C&#39;de Geltrex kaplı kültür kaplarına saklayabilir. Geltrex kurumasını önlemek için her tabak Parafilm Seal. </li><li> Kullanmadan önce, bir laminer akış kaputu Geltrex kaplı yemekler aktarmak ve onları oda sıcaklığında (yaklaşık 1 saat) gelmesini sağlar. </li></ol> Komple nakavt SR Besleyici Ücretsiz Orta hazırlanması <ol><li> 10 mcg / ml Temel FGF çözüm, 1 ml hazırlamak için, aseptik aşağıda listelenen bileşenleri karıştırın. -20 ° C&#39;de kısım çözüm ve mağaza, 6 aya kadar. <table border="0"><tbody><tr><td> Temel FGF </td><td> 10 mikrogram </td></tr><tr><td> D-PBS </td><td> 990 mcL </td></tr><tr><td> 10% BSA </td><td> 10 mcL </td></tr></tbody></table> Not: BSA aynı konsantrasyonda HSA veya Knockout SR ile ikame edilebilir. </li><li> 50 ml 2 mg / ml Dispase çözüm hazırlamak için, aseptik aşağıda listelenen bileşenleri karıştırın. Filtre 4 çözüm ve mağaza ° C 2 haftaya kadar sterilize. <table border="0"><tbody><tr><td> Dispase </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 ml </td></tr></tbody></table></li><li> 100 ml tam nakavt SR Besleyici-Free (KSR-FF) orta aseptik aşağıdaki tabloda listelenen bileşenleri bir araya hazırlamak için. <table border="1"><tbody><tr><td> Bileşen </td><td> Stok Konsantrasyon </td><td> Final Konsantrasyon </td><td> Hacim </td></tr><tr><td> Nakavt DMEM/F12 (Kat. No. 12.660-012) </td><td> - </td><td> 1X </td><td> 76.8 ml </td></tr><tr><td> GlutaMAX-I (Kat. No: 35.050-061) </td><td> 200 mm </td><td> 2 mM </td><td> 1 ml </td></tr><tr><td> Nakavt SR (Kat. no: 10.828-028) </td><td> - </td><td> % 20 </td><td> 20 ml </td></tr><tr><td> Nakavt SR-GFC (Kat. no. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 mL </td></tr><tr><td> bFGF (Kat.. PHG0024) </td><td> 10 mcg / mL </td><td> 20 ng / ml </td><td> 200 mcL </td></tr></tbody></table> Bir haftaya kadar 2-8 ° C&#39;de KSR-FF orta saklayabilir. </li><li> Sıcaklık ve gazlar için tam orta pre-değişikti hemen önce, aseptik 0.1 mM son konsantrasyon için gerekli 2 merkaptoetanol hacmi (55 mM stok konsantrasyonu) ekleyin. Örneğin, 100 ml KSR-FF orta hazırlamak için 182 mcL 2-mercaptoethanol (1:550 dilüsyon) Alternatif olarak 55 mM, 2-mercaptoethanol 1X tamamlanan orta ve 2-8 ° C&#39;de saklanan olabilir bir hafta kadar. </li></ol> KSR-FF Orta iPSCs uyarlanması <ol><li> Başlamadan önce, kaput Geltrex kaplı yemekleri oda sıcaklığında ön dengelenmesi. </li><li> Kültür MEF besleyici hücreler iPSCs% 70-80 konfluent kadar. MEF tabanlı kültür protokoller için Gibco Fare Embriyonik Fibroblast el kitabı bakın. </li&gt; <li> 37 ° C su banyosunda Dispase Öncesi sıcak gerekli hacim. Gerekli miktarlar hakkında ayrıntılı bilgi için aşağıdaki Tablo 1&#39;e bakınız. </li><li> 37 ° C su banyosunda for15 dakika KSR-FF gerekli hacmi Pre-dengelenmesi. Gerekli miktarlar hakkında ayrıntılı bilgi için aşağıdaki Tablo 1&#39;e bakınız. </li><li> Kültür yemekleri orta aspire ve uygun miktarda bir Dispase çözüm ekleyin. Bulaşıkları 3-5 dakika 37 ° C&#39;de inkübe edin. </li><li> Her kültürün gemi Dispase çözüm aspire ve D-PBS (2 ila 3 kat) MEF besleyici hücrelerin nazikçe yıkayın. </li><li> Her kültür gemi için uygun bir miktar tam nakavt SR orta ekleyin. Kapalı hücreleri kültür gemi yüzeyine hafifçe kazımak için bir hücre kazıyıcı ya da 5 ml pipet kullanın. Not: sabit iPS uyum hücre hatları için alternatif uyum protokoller için eke bakınız, </li><li> Her kültürün çanak hücre süspansiyonu ayrı 15 ml konik tüpler içine toplayın. Her bir kültür damarı tam nakavt SR orta uygun bir miktarı ile temizleyin, durulayın ve D-PBS hücre süspansiyonu içeren 15 ml konik tüpler içine orta durulama ekleyin. Tek hücre içine hücre kümeleri kırmak için değil dikkatli olun </li><li> 15 ml konik tüpler iPSCs pelet 5 dakika boyunca 200 xg&#39;de santrifüjleyin. </li><li> IPSC pelet süpernatant aspire. KSR-FF orta split oranı (Tablo 1) göre uygun bir miktarda pelletini tekrar. Küçük öbekler yüzeye iyi binmemelidir, çünkü küçük bir boyutu, hücre kümeleri kırmak değil. Not: Biz ilk 3 pasajlar iPSCs iPSC MEF Kültür Orta KSR-FF orta direkt geçişli olmuştur sonra 1:2 bölünmüş bir oranı öneririz. Normalde, 01:03 ve 01:05 arasında bir bölünme oranı uygun olan, ancak 01:02 az Pasajlanması besleyici ücretsiz bir kültürün içine adapte gerekli hücrelerinin yüksek yoğunluklu sağlar. </li><li> Geltrex kaplı yemekleri iPSCs kaplama, önce ön kaplı çanak artık Geltrex çözümü aspirat ve yavaş yavaş her kültür çanak için uygun bir miktarda hücre süspansiyonu ekleyin. Not: kaplama önce yemekleri durulama yok. </li><li> Çanağı yüzeyi boyunca hücreler dağıtmak için birkaç kez ileri geri ve yan kültür çanak hareket ettirin. Hafifçe nemlendirilmiş bir atmosfer hava 4 ila% 6 CO 2 ile 37 ° C inkübatör kültür tabağına yerleştirin. KSR-FF ile harcanan orta her gün değiştirin. </li></ol> KSR-FF kullanarak Pasajlanması insan iPS hücreleri <ol start="13"><li> Mikroskop altında hücrelerin% 70-80 konfluent ve pasajlandı olmaya hazır olduğunu doğrulamak için tam KSR-FF büyüyen insan iPSCs uyun. Şekil 1 bakın. Not: koloniler çok yoğun veya çok büyük hale gelirse, artan farklılaşması oluşur. </li><li> Kes ve kültür Pasajlanması önce herhangi bir farklılaşmış iPSC koloniler kaldırmak. </li><li> 37 ° C su banyosunda Dispase Öncesi sıcak gerekli hacim. Gerekli miktarlar hakkında ayrıntılı bilgi için aşağıdaki Tablo 1&#39;e bakınız. </li><li> 37 ° C su banyosunda for15 dakika KSR-FF gerekli hacmi Pre-dengelenmesi. Gerekli miktarlar hakkında ayrıntılı bilgi için aşağıdaki Tablo 1&#39;e bakınız. </li><li> Bir pipet kullanarak kültür damarı harcanan orta aspire ve hücrelerin D-PBS ile iki kez yıkayın. </li><li> Yavaşça kültür damarı (örneğin, 60 mm kültür çanak başına Dispase çözüm 1 ml) önceden ısıtılmış Dispase çözüm ekleyin. Tüm hücre yüzeyinde kat Swirl kültür damarı. </li><li> Kültür damarı 3 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Inkübatör geminin çıkarın, Dispase çözüm aspirat ve D-PBS ile hücrelerin nazikçe yıkayın. </li><li> Hücrelerin, hücre kazıyıcı kullanarak kültürü çanak yüzeyinden hafifçe kazıyın ve 15 mL steril bir santrifüj tüpüne hücreleri aktarmak. </li><li> Nazikçe müstakil olmayan herhangi bir hücre &quot;püskürtme&quot;, KSR-FF ile iki kez kültür çanak durulayın Havuz, 15 mL tüp hücreleri orta durulayın. </li><li> Tüp, oda sıcaklığında pelet hücreleri 5 dakika boyunca 200 xg&#39;de santrifüjleyin . </li><li> Dikkatle hücre pelletini bozmadan süpernatantı aspirat ve atın. </li><li> Yavaşça tüp tüp alttan tamamen hücre pelletini çıkarmak için hafifçe vurun. </li><li> 5ml serolojik bir pipet kullanarak ön dengelenmiş KSR-FF hücreler yavaşça tekrar süspansiyon haline getirin. Çiğnemek değil. Not: hafifçe zarar vermemek için kuvvet kullanmadan hücreleri tekrar süspansiyon adım kritik. </li><li> Istenen bölünmüş oranında taze bir 60 mm Geltrex kaplı çanak hücreleri transferi ve yüzeyi boyunca hücreler dağıtmak için birkaç kez ileri geri ve yan kültür çanak taşımak. </li><li> Nemlendirilmiş bir atmosferi ile 37 ° C inkübatör kültür tabağına yerleştirin% 4 ila 6 hava CO 2. </li><li> Ertesi gün hafifçe hücre enkaz kaldırmak için harcanan orta KSR-FF ile değiştirin. Bundan sonra günlük harcanan orta değiştirin. IPS hücreleri günlük olarak (yaklaşık olarak her 4-5 gün) geçişi dikkate alınmalıdır. Pasajlanması hücrelerin% 70-80 izdiham ulaştığınızda tavsiye edilir. IPS hücre dondurulması ve eritme için, &quot;Komple nakavt Serum Yedek Besleyici-Alerjik Orta kullanarak dondurularak ve İnsan iPS Hücreler kurtarma&quot; başlıklı protokol bakın. </li></ol> Beklenen Sonuçlar <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Şekil 1" /> Şekil 1. Aşağıdaki faz kontrast görüntü tam nakavt Serum Yedek Besleyici Ücretsiz orta kullanarak Geltrex kaplı kültür kaplarına yetişen iPSCs gösterir. IPSCs, büyük bir çekirdek ve kıt sitoplazma ile karakterize hESC, morfolojisi benzer sergiler. (40x büyütme) <table border="1"><tbody><tr><td> Bileşen </td><td> 35mm Bulaşık </td><td> 60mm Bulaşık </td><td> 100mm Bulaşık </td></tr><tr valign="top"><td> Komple nakavt SR orta </td><td> 2 mL </td><td> 4 mL </td><td> 10 ml </td></tr><tr valign="top"><td> Geltrex Çözüm </td><td> 1 ml </td><td> 1,5 mL </td><td> 4-5 ml </td></tr><tr valign="top"><td> Dispase </td><td> 0,5 mL </td><td> 1 ml </td><td> 3-4 ml </td></tr><tr valign="top"><td> Durulama için D-PBS </td><td> 2 mL </td><td> 4 mL </td><td> 10 ml </td></tr></tbody></table> Tablo 1. Önerilen Hacimleri <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/2236/2236appendix.pdf">Ek görmek için buraya tıklayınız</a> .

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	<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <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>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <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+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <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+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, 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=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <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.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

Bu makalenin yazarları, bu yazıda kullanılan reaktifler ve aletleri üreten Life Teknolojileri tarafından istihdam edilmektedir.

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>Cat. no. 12660-012</td><td>Note: see appendix for the use of alternative DMEM products</td></tr><tr><td>GlutaMAX-I</td><td></td><td>Cat. No. 35050-061</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>KnockOut SR, Cat. no. 10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>KnockOut SR-GFC, Cat. no. A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>bFGF, Cat. no. PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>Cat. no. 21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>Cat. no. 17105-041</td><td>Note: see appendix for alternative passaging methods.</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>Cat. no. A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td><td></td><td>Cat. no. 14190-144</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr></tbody></table>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

2006 yılında, insan ve fare fibroblastlar dikkat çekici embriyonik kök hücrelere benzer nitelikleri ile 1-3 iPS hücreleri üretmek yeniden olabilir keşif pluripotent hücrelerin hücre tedavisi, ilaç keşfi ve temel araştırmalar için değerli bir yeni kaynak yaratmıştır . Gibco medya ve reaktifler yıllardır pluripotent kök hücre araştırmaları ön planda olmuştur. DMEM Nakavt Nakavt Serum Değiştirme ile desteklenmiş, embriyonik kök hücre büyümesi ve şimdi iPS hücre kültürü 3-9 için tercih edilen medya . Bu altın standart medya sistemi artık Nakavt SR Büyüme Faktörü Kokteyli ilavesi ile besleyici ücretsiz kültür kullanılabilir. Geleneksel insan ES ve iPS hücre kültürü yöntemleri, fare veya insan fibroblast besleyici katmanları veya besleyici klimalı ortamı gerektirir. Bu kültür yöntemleri ölçekli, emek-yoğun, sert ve tanımlanmamış durumlar nedeniyle farklılaşmamış kalça hücreleri korumak için zordur. Invitrogen Nakavt SR hala korurken, besleyici-ücretsiz geçiş kalça hücre kültürlerinde kolaylıkla izin Nakavt SR Büyüme Faktörü Kokteyl geliştirdi.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Aşağıdaki protokolü tam nakavt Serum Yedek Besleyici Ücretsiz orta (KSR-FF) kullanarak besleyici ücretsiz kültür, insan kaynaklı Pluripotent Kök (IPS) Hücreler uyarlanması için talimat sağlar. Bir kez, adapte sürekli bakım için talimatlar ayrıca verilmiştir.

İnsan IPS Hücreler Komple nakavt Serum Yedek Besleyici-Alerjik Orta kullanarak Besleyici Adaptasyon, Kültür ve Pasajlanması

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

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Universidad San Sebastian

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JoVE Journal

Biology

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Video: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

Rutin İmmünositokimyasal Karakterizasyonu için serum serbest şartlar İnsan iPSCs küçük ölçekli Yayılım

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

Gelişim Biyolojisi

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Tanımlanmış Xeno-alerjik ve besleyici-Alerjik kültür koşulları üretimi insan IPSC kaynaklı Retina hücre modelleri için

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Somite türevleri indüklenen insan nesil Vitro Pluripotent kök hücreler

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Nörobilim

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

İnsan iPS hücreleri dondurularak ve kurtarma Tam nakavt Serum Yedek Besleyici-Alerjik Orta

Biyoloji

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 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 iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Epizomlar ve HDAC inhibitörleri kullanarak Blood Verimli iPS Hücre Üretimi

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Kimerik Antijen Reseptörü İfade Eden Doğal Öldürücü Hücrelerdeki Gelişmeler

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...

İmmünoloji ve Enfeksiyon

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells&#39; differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures.
To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting).
In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for&#160;colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

To avoid the limitations associated with the enzymatic or mechanical passaging of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) cultured on feeder cells, we have established a fast, effective, cost-efficient, high-yield method for harvesting hESC or hiPSC colonies maintained on a feeder cell layer of human foreskin fibroblasts using EDTA-mediated dis-adhesion.

İnsan pluripotent kök hücrelerinin besleyici hücreler üzerinde etilendiamintetraasetik asit aracılı yapışma ile hızlı, uygun maliyetli, enzimsiz geçişi

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different ...