Name: feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium
Uploaded: 2010-07-15T17:00:00.000+00:00
Duration: 8 min 29 s
Description: 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

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

Research

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

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel Kültür Modeli İnsan Pluripotent için Plasenta klimalı Medyada Jelatin üzerinde hücre çoğalma Kök

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Gelişim Biyolojisi

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

İnsan Pluripotent Kök Hücreleri dopaminerjik Nöron Farklılaşma Yönetmen

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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

Otomatik Kültür Sistemi Kullanılarak İndüklenmiş Pluripotent Kök Hücre Farklılaşması

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Yazar Spotlight: Gelişmiş Tekrarlanabilirlik için iPSC Kültürünü Otomatikleştirme 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

İnsan kaynaklı pluripotent kök hücrelerin korunması ve ayırt edilmesi için otomatik bir kültür sistemi

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Yüksek Yoğunluklu İnsan Embriyonik Kök Hücre Kültürleri gelen Rapid Fibroblast Kaldırma

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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

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