1. Reprogramming <ol><li> 6 plaka başına 1 x 10 5 hücre yoğunluğu Tohum BJ hücreleri. Kültür hücrelerinin büyüme orta 2 ml (EMEM 450 ml, 50 ml ES nitelikli FBS, 5 ml 10 mM non-esansiyel amino asit, 5 ml penisilin / streptomisin, 5 ml 200 mM L-glutamin ve 0.9 ml 55 mM β ile desteklenmiş 37 o gecede merkaptoetanol) ° C ve% 5 CO 2. </li><li> Aynı gün, 6 plaka su içinde seyreltilmesi jelatin% 0.1 ve 37 geceleme · C ve% 5 CO 2 kuluçka ekleyerek MEF besleyici plakaları hazırlanmaya başlayacak. </li><li> Inkübasyondan sonra, BJ hücrelerden orta kaldırmak ve her iyi polybrene 6 mikrogram / ml tamamlanan 2 ml besi, 500 ul hOct4 lentivirüs, 50 ul hSox2 lentivirüs, 50 ul hNanog lentivirüs ve 50 ul hLin28 lentivirüs ekleyin . Orta hatta kaplama sağlamak için plaka yavaşça sallayın. 37 ° ° C ve% 5 CO 2 gece inkübe. </li><li> Aynı gün, 1 şişe sıvı azot ve çözme CF-1 MEF hücrelerinin çıkarın. Jelatin kaplı kuyular (adım 1.2 &#39;ye bakınız) sıvı çıkarın ve 0,2 x 10 5 hücre / iyi bir yoğunluk MEF besleyici hücreler ekleyin. MEF büyüme orta (450 ml DMEM 50 ml FBS ve 5 ml non-esansiyel amino asitleri ile takviye) yanı başına toplam hacmi hücre 2ml olmalıdır. </li><li> Ertesi gün, 5 dakika boyunca 200 xg&#39;de 0.05% tripsin / EDTA ve santrifüj BJ hücreleri ayırın. Büyüme orta, orta ve tekrar süspansiyon aspire. MEF besleyici plaka (adım 1.4 &#39;e bakınız), orta çıkarın ve 2 ml / BJ hücre süspansiyonu ekleyin. Hücre konsantrasyonu yaklaşık 5 x 10 4 hücre / iyi olmalıdır. 37 ° ° C ve% 5 CO 2 gece inkübe. </li><li> Orta 24 saat yeniden tohumlama sonra insan ES / iPS hücre kültürü, orta (DMEM/F12 400 ml, 100 ml Nakavt serum Yedek, 5 ml 10 mM non-esansiyel amino asit, 5 ml 200 mM L-glutamin, 0.9 ml ile takviye ile değiştirin 55 mM β-mercaptoethanol ve 20 ng / ml insan rekombinant bFGF). 7 gün boyunca her 24 saatte bir, orta değiştirin. </li><li> Yedi gün sonra, MEF klimalı ortamda orta değiştirme (bkz. Bölüm 2). </li></ol> 2. MEF Koşullu Orta hazırlanması <ol><li> Tohum CF-1, 2 x 10 5 hücre MEF besleyici hücreler / 2 ml MEF büyüme ortamında 6 plaka üzerinde . 37 ° ° C ve% 5 CO 2 gece inkübe. </li><li> Orta ve insan ES / iPS hücre kültür ortamı değiştirin. 37 ° ° C ve% 5 CO 2 gece inkübe. </li><li> Dört gün boyunca her 24 saatte bir süpernatant toplayın. 0.22 mikron filtre ile süpernatantı Filtresi. </li><li> BFGF 50 ng / ml supernatant Ek. </li></ol> 3. iPS Koloni Seçimi ve Pasajlanması <ol><li> CF-1 MEF besleyici hücreler ile hücre kültür yemekleri önceden numaralı seribaşı Stemolecule Y27632 10 mcM ile desteklenmiş insan ES / iPS kültür ortamı ES-benzeri bir koloni ve yeniden tohum seçin. 37 ° ° C ve% 5 CO 2 gece inkübe. </li><li> İlk yedi gün (Stemolecule Y27632 ile tamamlayan olmadan) için her 24 saatte bir, orta kültür değiştirin. Yedi gün sonra, MEF klimalı ortamda orta değiştirmek (hazırlık için bölüm 2&#39;ye bakınız). Tipik bir insan ES morfolojisi gösterdi kadar hücreleri Pasajlanması devam etti. </li></ol> 4. Pluripotency Belirteçleri İmmünositokimyasal Sınav <ol><li> Hücreler PBS ile hafifçe üç kez yıkayın. </li><li> 500 oda sıcaklığında 20 dakika fiksatif ul hücreleri sabitleyin. </li><li> Hücreler PBS ile hafifçe üç kez yıkayın. </li><li> Oda sıcaklığında bir saat için 500 ul engelleme tampon ile non-spesifik bağlama engelleyin. </li><li> 4 geceleme birincil antikor ile 250 ul hücreleri ° C&#39;de (1:100 TRA-1-81, TRA-1-60, SSEA-4, SSEA-3, Oct4, Sox2, Nanog ve Lin28 dilüsyonları). </li><li> Hücreler PBS ile hafifçe üç kez yıkayın. </li><li> (Anti-fare IgG Cy3 konjuge, Keçi anti-fare IgG Cy3 konjuge keçi anti-Sıçan IgM Cy3 konjuge ve keçi anti-Keçi kullanılan ışıktan uzak tutarak, hücrelerin oda sıcaklığında 1 saat süreyle 250 ul ikincil antikor ile inkübe Tavşan Cy3 konjuge) 1:300 dilüsyonları. </li><li> Hücreler PBS ile hafifçe üç kez yıkayın. </li><li> Son yıkama DAPI (son konsantrasyonu 1 mcg / ml) ve çekirdekleri görselleştirmek için 5 dakika inkübe edin. </li><li> Büyütme altında hücrelerin analiz edin. </li></ol> Bölüm 5: Temsilci Sonuçlar 1. Morfoloji Sonuçlar: İnsan sünnet derisi fibroblast (BJ) hücreleri, Sox2, Nanog ve Lin28 Oct4 birlikte transduced. Günde 4 post-transdüksiyon gibi erken morfolojik değişiklikler gözlenir ve hücre kümesi daha sıkı gün 17 (Şekil 1) dolu oldu. Kolonileri 25 gün elle aldı ve CF-1 MEF besleyici hücreler kültüre edildi. IPS hücrenin yeniden programlanması sonra koloni oluşumunu kolaylaştırmak için, biz Stemolecule Y27632ROCK inhibitörü, her geçiş sırasında ilk gecede tohumlama için. iyi morfolojisi iPS hücre kolonileri koloni üç sıralı mermi toplama ve Pasajlanması sonra gözlendi. 2. Pluripotency Markers İfade: Izole iPS hücre kolonileri karakterize etmek için, biz ES hücrelerinin ifade ortak pluripotency belirteçlerin varlığı için baktı. Koloniler güçlü alkalin fosfataz (AP) aktivitesi (Şekil 2) sergiledi. Ayrıca, immünsitokimya (ICC) yüzey belirteçleri TRA-1-81, TRA-1-60, SSEA-4 ve SSEA-3 gibi nükleer belirteçlerinin de dahil olmak üzere, pluripotency marker spesifik antikorların bir panel ile iPS hücre kolonileri yapıldı Oct4, Sox2 ve Nanog. Izole iPS koloniler Tüm göstergeler için pozitif (Şekil 3). ICC sonuçları iPS hücreleri bu iPS hücreleri farklılaşmamış insan ES hücreleri yakından benzediği gösteren uygun pluripotency işaretleyici ifade desenini ortaya koyduğu göstermektedir. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Şekil 1" /> Şekil 1: Morfolojik değişiklikler BJ hücreleri transduced. Aydınlık alan görüntüleri, tipik bir iPS hücre kolonisinin (A) 4 gün ve (B) 17 gün sonrası transdüksiyon kurdu. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Şekil 2" /> Şekil 2: AP reprogrammed BJ hücreleri aktivite. Üç farklı koloniler Stemgent Alkalin Fosfataz Boyama Seti ile boyandı. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Şekil 3" /> Şekil 3: yüzey belirteçleri TRA-1-81, TRA-1-60, SSEA-4 ve SSEA-3, ve nükleer belirteçler Eki 4, Nanog ve Sox2: İnsan iPS hücreleri aşağıdaki ES hücre spesifik belirteçler yüksek düzeyde ifade.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, 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=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 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

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

Bu sonuçlar, insan fibroblastlarında transkripsiyon faktörleri transduced Stemgent İnsan TF lentivirüs Set verimli ektopik ifade uyararak iPS koloniler oluşturmak için kullanılabilir olduğunu göstermektedir. Reprogramming deneyler tasarlarken, çok değişkenli reprogramming verimliliği optimize etmek için düşünülmelidir. İlk olarak, aktif virüs hedef oranı (enfeksiyon, İçişleri Bakanlığı, çokluk) optimum transduction verim elde etmek için birincil transdüksiyon adım sırasında değiştirilmesi gerekir. İkinci olarak, hedef hücrelerin büyüme durumu yeniden programlama etkileyebilir. Sağlıklı ve proliferatif hücreleri yeniden programlama için daha müsait. Üçüncü olarak, farklı hücre numaraları için protokol değiştirerek, hedef hücre numaraları kültür çanak yüzey alanı ile orantılı ayarlanabilir olması tavsiye edilir. Son olarak, son çalışmaların artırılması embriyonik kök koloni hayatta kalma kendi programını gösterdiği gibi, başarılı bir yeniden programlama yardımcı olmak için ROCK inhibitörleri gibi Y27632 başvurusu dikkate alınması gerekir. 4,5

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>00-0005</td>
<td>&nbsp;</td>
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<td>Stemolecule Y27632</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0012</td>
<td>&nbsp;</td>
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<td>TRA-1-81 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0012</td>
<td>&nbsp;</td>
<tr>
<td>TRA-1-60 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0009</td>
<td>&nbsp;</td>
<tr>
<td>SSEA-4 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0003</td>
<td>&nbsp;</td>
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<td>SSEA-3 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

Yamanaka ve arkadaşları 2006 yılında, ilk Oct4, retrovirüs aracılı teslimat ve ifade, Sox2, c-Myc ve Klf4 fare fibroblastlar pluripotent devlet tetikleme kapasitesine sahip olduğunu göstermiştir. 1 aynı grup aynı zamanda insan somatik hücre başarılı bir şekilde yeniden programlanması halinde rapor başka bir dizi faktöre lentivirüs aracılı co-ifade (Oct4, Sox2, Nanog ve Lin28 retroviral vektörler aynı transkripsiyon faktörleri tarafından teslim insan sürümlerini kullanan bağlı pluripotent kök (iPS) hücreleri, 2 Ayrıca, James Thomson ve ark. ) iPS hücreleri içine insan somatik hücre yeniden programlama yeteneğine sahip olan 3 iPS hücreleri morfolojisi, çoğalması ve vücudun tüm doku tiplerine ayırt etme yeteneği ES hücrelere benzer. İnsan iPS hücreleri embriyo imha etik ikilem olmadan ES hücrelerinin temel özellikleri sergilerler ES hücreleri üzerinde ayrı bir avantajı var. Hastaya özgü iPS hücreleri üretimi, kişiselleştirilmiş rejeneratif tıp tedavilere olmayan otolog nakledilen hücrelerin bağışıklık ret potansiyeli ortadan kaldırarak önemli bir barikat circumvents. Burada Stemgent İnsan TF lentivirüs Set kullanarak insan fibroblast hücreleri yeniden programlama için protokol göstermektedir. Ayrıca bu set ile reprogrammed hücreleri morfolojisi dört gün sonrası transdüksiyon iPS göstermeye başladığını göstermektedir. Stemolecule Y27632 kullanarak, toplama ve koloni üç sıralı mermi Pasajlanması sonra iPS hücreleri ve gözlenen doğru morfolojisi için seçti. Biz de göstermektedir ki hücreleri yeniden programlama pluripotency işaretleyici AP, yüzey belirteçleri TRA-1-81, TRA-1-60, SSEA-4 ve SSEA-3, ve nükleer belirteçler Oct4, Sox2 ve Nanog görüntülenir.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

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

Biz, insan faktörleri Oct4, Sox2, Nanog ve Lin28 lentivirüs aracılı teslim kullanarak insan somatik hücre alınan uyarılmış pluripotent kök hücreleri üretimi için protokol göstermektedir. Pluripotency morfolojisi ve embriyonik kök (ES) hücre-spesifik belirteçler varlığı teyit edilmiştir.

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 ...

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Unit 1

Embryonic and Induced Pluripotent Stem Cells

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29.8K Görüntüleme.  Stemgent. Biz, insan faktörleri Oct4, Sox2, Nanog ve Lin28 lentivirüs aracılı teslim kullanarak insan somatik hücre alınan uyarılmış pluripotent kök hücreleri üretimi için protokol göstermektedir. Pluripotency morfolojisi ve embriyonik kök (ES) hücre-spesifik belirteçler varlığı teyit edilmiştir.

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biyoloji

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

İnsan Embriyonik Kök Hücre Kültürü ve Bakım

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Dört Transkripsiyon Faktör Fare Embriyonik Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi, Doksisiklin lentiviral İletim Sistemi Đndüklenebilir

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

İnsan Embriyonik Kök Hücreler Donma ve Çözülme

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Biyogüvenlik Geliştirilmiş Polimer complexed Ecotropic lentivirüs İnsan Hücreleri, İletimi

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

İnsan İsteyerek Pluripotent Kök Hücreler transfekte ve Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

İnsan İsteyerek Pluripotent Kök Hücre seçme ve ayırma Kolonileri Yetişkin Fibroblastlar gelen reprogrammed

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP Retroviral Vector kullanarak İsteyerek Pluripotent Kök Hücrelerinin (iPSCs) içine İnsan somatik hücreleri yeniden programlama

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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

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

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

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

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

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