Bu eser Charles Hood Vakfı Tıp ve Çocuk Sağlığı Araştırma Ödülü, Yale School of tarafından finanse edildi.

1. Yeniden programlanması Faktörler ifade Retrovirus tarafından yeniden programlama <ol><li> İnsan fibroblastlar fibroblast ortamı (10% Pen / Strep ile DMEM içinde FBS) kültürlenmiştir. </li><li> 6-iyi plaka iyi birine enfeksiyonu bir gün önce, plaka 1x10 5 insan fibroblastları. </li><li> Ölü hücreleri çıkarmak ve taze fibroblast orta 2 ml eklemek için orta aspire. 5 ug / ml &#39;lik nihai bir konsantrasyon protamin sülfat ekleyin. </li><li> Dikkatli bir enfeksiyon çokluğu (İçişleri Bakanlığı) 5 5 karşılık gelen her GFP-ifade virüs uygun miktarda ekleyin. </li><li> 2 ml fibroblast orta ekleyin, bir gün sonra enfeksiyon, viral süpernatant kaldırmak 2 ml PBS ile üç kez yıkayın. </li><li> Üç gün enfeksiyondan sonra, GFP flöresanı kontrol etmek ve 2 ml fibroblast ortam ile de takviye. </li><li> Bir üzerine fibroblast ortamda dört gün enfeksiyondan sonra, plaka 1x10 ışınlanmış fare embriyonik fibroblast 4 / cm 2 (MEFS) besleyici hücreler10-santimetrelik bir petri tabağına% 0.1 jelatin ile kaplanır. 37 ° C&#39;da gece boyunca inkübe edilir. </li><li> Beş gün enfeksiyondan sonra, 1 ml% 0.05 typsin / EDTA 5 dakika boyunca 37 ° C&#39;de ve 200 g hızında 5 dakika boyunca santrifüj ile enfekte insan fibroblastlar ayırmak. Ortamı aspire ve fibroblast orta 10ml ile tekrar süspansiyon hücreleri. Bir ön-kaplamalı 10 cm-plakası içine hücreleri transfer edin. </li><li> 24 saat sonra, hESC kültür ortamı (% 20 Knock-Out serum yerine, DMEM/F12, 0.1 ml Esansiyel olmayan amino asitler, 4 ng / ml bFGF, Pen / Strep / Glutamat, beta-merceptoethanol) ile orta yerine. Günlük orta değiştirin. ESC gibi koloniler enfeksiyondan sonra günde 20-27 tarafından görünmeye başlayacaktır. </li></ol> 2. IPSCs izolasyonu ve Genişletme <ol><li> Bir floresan mikroskop altında, hESC benzer bir morfoloji gösteren bir kolonide GFP floresans olmaması için kontrol edin. </li><li> Bir 10 ul pipet kullanarak, bireysel iPSC koloniler almak ve bir jelatin ve MEF-COA iyi birine koyunted 12-iyi plaka ve hESC orta ile desteklenmiş. Günlük orta değiştirin. </li><li> Pasajlanmasını için, DMEM/F12 1 ml yıkama plakası, daha sonra kollajenaz 0.5 mL, 37 ve 10 dakika boyunca kuluçkaya ° C. </li><li> DMEM/F12 ile iki kez yıkanır hücreleri. </li><li> Taze hESC orta 2 ml ekleyin. Bir hücre hırsızı kullanarak, küçük parçalar halinde koloniler parçalamak ve plakadan kalan hücreler ayırmak. </li><li> Jelatin ve MEF kaplı 6-iyi plaka iyi birine kolonilerin süspanse adet aktarın. </li></ol> 3. Pluripotent Markers immünofloresan Analizi <ol><li> PBS ile üç kez yıkanır hücreleri ve oda sıcaklığında 20 dakika için% 4 paraformaldehit ile sabitlemek. </li><li> Yavaşça ile% 0.2 Triton 30 dakika süreyle PBS içinde X-100. PBS ile üç kez hücreleri yıkayıp geçirgenliği </li><li> Iki saat süreyle PBS içinde% 3 BSA ile kuluçkaya hücreleri tarafından spesifik olmayan bağlanma bloke eder. </li><li> 4 &#39;de bir gece primer antikor ile birlikte inkübe hücreleri ° C. </li> &lt;li&gt; PBS ile üç kez yıkanır hücreleri ve ışıktan koruyucu, oda sıcaklığında bir saat boyunca belirli bir sekonder antikor ile hücreleri inkübe edilir. </li><li> 5 dakika boyunca oda sıcaklığında inkübe edildi, son yıkama sırasında PBS ve eklentinin DAPI ile hücreleri, üç kez yıkanır. </li><li> Bir floresan mikroskop ile boyama algılar. </li></ol> 4. Pluripotent Markers için Kantitatif Real-time PCR <ol><li> Qiagen Kullanıcı RNeasy kiti kullanılarak, insan fibroblastlarının türetilmiş insan iPSCs toplam RNA izole edin. </li><li> Üst Simge II Reverse Transcriptase kullanarak ilk iplikli cDNA sentez. </li><li> Daha önce rapor primerler kullanılarak pluripotency genleri tespit etmek qPCR gerçekleştirin. 6 </li></ol> 5.. Temsilcisi Sonuçlar <ol><li> Yeniden programlama sırasında Morfolojik değişim Biz OCT4, SOX2, KLF4 ve MYC taşıyan retrovirüslerin bir kokteyl ile insan fibroblastları BJ1 ve Detroit 551 enfekteve (Şekil 1) yeniden programlama sırasında morfolojik değişiklikler tespit edebildi. Yirmi bir gün enfeksiyondan sonra, onların hESC benzeri morfolojisi ile küçük iPSC koloniler farkındayız. Ayrıca, GFP floresan iPSCs farkındayız. Böyle EKH ve iPSCs olarak pluripotent kök hücreler, proviral gen 7-9 bastırmak için moleküler makineler ifade. Bizim eşsiz retroviral vektörü retroviral LTR genleri yeniden programlama ile birlikte GFP ifade eder. Böylece sürekli GFP salgılayan hücrelerin proviral gen suskunluğu olmadan transgenlerin ifade etmek için kabul edilir. Pluripotency moleküler ağ elde Sadakatle reprogrammed iPSC koloniler GFP (Şekil 2) 10 olmadığını göstermektedir. </li><li> Insan iPSCs ve pluripotency Karakterizasyonu Biz Tra-1-81, Tra-1-60, SSEA-4, SSEA-3, OCT4 ve NANOG antikorlar (imünohistokimya ile Detroit-551 fibroblastlar türetilen koloniler analiz <strong&gt; Tablo 2). Başarıyla yeniden programlanması iPSCs bu belirteçler (Şekil 3A) tüm ifade. Ayrıca, kantitatif RT-PCR analizi ile gen ifadesi analiz edilmiştir. Biz OCT4 ifadesi, SOX2, KLF4, MYC ve NANOG anlamlı ebeveyn fibroblast hücreleri ile karşılaştırıldığında artmış ancak H9 hESC (Şekil 3B) bu ile orantılı olduğu görülmektedir. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/3804/3804fig1.jpg" /> Şekil 1.. Retrovirüs-enfekte insan fibroblastları morfolojik değişiklikler. (AD) yeniden programlama faktörleri ile enfekte Detroit-551 fibroblastlardan koloniler Progressive morfolojik değişim. Gün 5 (A), 10 gün (B), Günlük 14 (C), günde 21 (D). Hücreler 21 gün sonra hESC benzeri morfoloji göstermektedir. <img alt="Şekil 2" src="/files/ftp_upload/3804/3804fig2.jpg" /> Şekil 2. Reprogramming giren hücrelerde Temsilcisi GFP floresan ifade. </strong&gt; BJ ve Detroit 551 fibroblastlar retrovirüs dört yeniden programlama faktörleri 4 ifade ile enfekte ve dört hafta boyunca hESC ortamda inkübe edildi. BJ fibroblast (A, B) ve Detroit 551 (C, D) morfolojik benzer göstermektedir. Günlük 21, GFP negatif koloniler iyi niyetli iPSCs 10 temsil eden oluşturmaya başlar. (E, F) uygun reprogramming uğramamış Detroit 551 hücreler dönüştürdü göstermektedir. Faz kontrast görünümü altında (A, C, E) koloniler. (B, D) düzgün GFP susturulması göstermek hücrelerin yeniden programlanması. Transforme koloni (F) parlak GFP. <img alt="Şekil 3" src="/files/ftp_upload/3804/3804fig3.jpg" /> Şekil 3. Insan kaynaklı pluripotent kök hücrelerin karakterizasyonu. (A) İnsan 551-iPS-K1 hücre kolonilerinin pluripotent hücreler için ortak işaretleri ifade. DAPI boyama alan başına toplam hücre içeriğini gösterir. Ebeveyn f (B) Kantitatif gerçek OCT4 ifade için zaman-PCR (RT-qPCR), SOX2, KLF4, MYCibroblast, 551-iPS-K1 iPSCs ve H9 insan embriyonik kök hücreleri (hESC). Veri β-aktin housekeeping gen karşı normalize ve ebeveyn fibroblast hücreleri 4 ifadesi seviyesine göre çizildi.

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<p class="jove_content"&gt; Biz ifşa hiçbir şey yok.</p

IPSCs dört transkripsiyon faktörleri reprograms insan fibroblast Anlatım. Pek çok klinik deneme güvenli iPSCs üretmek için non-entegre olan ya da olmayan genetik yaklaşım kullanılarak insan iPSCs üretmek için yapılmıştır. Şimdiye kadar, bu yöntemler son derece düşük verimlilik göstermek ve tekrarlanabilirlik 11-14 geliştirmek için daha fazla optimizasyon gerektirir. Retro-ya lentiviral yöntemlerle kolayca elde ve viral entegrasyon kaynaklanan sorunları emniyet az bağımlı olan ...

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">hESC medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>80%</td>
			<td>Invitrogen</td>
			<td>11330057</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>20%</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>2 mM</td>
			<td>Invitrogen</td>
			<td>25030081</td>
		</tr>
		<tr>
			<td>Nonessential Amino Acids (10 mM)</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>11140050</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol (14.3 M) or MTG</td>
			<td>0.1 mM</td>
			<td>Invitrogen</td>
			<td>M-6250</td>
		</tr>
		<tr>
			<td>bFGF-2 10 &mu;g/ml</td>
			<td>4 ng/ml</td>
			<td>GIBCO/BRL</td>
			<td>GF003AF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td colspan="4">Fiboblasts Medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>90%</td>
			<td>Invitrogen</td>
			<td>11965118</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
			<td>Invitrogen</td>
			<td>10407028</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>Millipore</td>
			<td>15140-122</td>
		</tr>
	</tbody>
</table>
Table 1. Culture Medium
<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Concentration</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab19857</td>
		</tr>
		<tr>
			<td>SSEA3</td>
			<td>1:100</td>
			<td>Milipore</td>
			<td>MAB4303</td>
		</tr>
		<tr>
			<td>SSEA4</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560218</td>
		</tr>
		<tr>
			<td>Tra-1-81</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560173</td>
		</tr>
		<tr>
			<td>Tra-1-60</td>
			<td>1:100</td>
			<td>BD Biosciences</td>
			<td>BD560174</td>
		</tr>
		<tr>
			<td>NANOG</td>
			<td>1:500</td>
			<td>Abcam</td>
			<td>Ab21624</td>
		</tr>
		<tr>
			<td>Alexa-Flur 488</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa-Flur 555</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:5000</td>
			<td>Invitrogen</td>
			<td>D1306</td>
		</tr>
		<tr>
			<td colspan="4">Plasmids</td>
		</tr>
		<tr>
			<td>pMIG-OCT4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17225</td>
		</tr>
		<tr>
			<td>pMIG-SOX2</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17226</td>
		</tr>
		<tr>
			<td>pMIG-KLF4</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>17227</td>
		</tr>
		<tr>
			<td>pMIG-MYC</td>
			<td>&nbsp;</td>
			<td>Addgene</td>
			<td>18119</td>
		</tr>
		<tr>
			<td colspan="4">Other Materials</td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>1mg/ml</td>
			<td>Invitrogen</td>
			<td>17104019</td>
		</tr>
		<tr>
			<td>Gelatin, Porcine</td>
			<td>0.1%</td>
			<td>Sigma</td>
			<td>G 1890</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>0.2%</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>4%</td>
			<td>Sigma</td>
			<td>47608</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>3%</td>
			<td>American Bioanalytical</td>
			<td>AB01800</td>
		</tr>
		<tr>
			<td>MEF feeder cells</td>
			<td>&nbsp;</td>
			<td>Millipore</td>
			<td>PMEF-N</td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>&nbsp;</td>
			<td>Corning</td>
			<td>3008</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td colspan="4">Fluorescent microscopy: inverted microscope with GFP filter</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and equipment.

reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp

İnsan embriyonik kök hücreleri (hESC) pluripotent ve in vitro hastalığı modelleme ve rejeneratif tıp 1 de için paha biçilmez bir hücresel kaynaklarıdır. Daha önce insan somatik hücre dört transkripsiyon faktörlerinin ektopik ifade (Oct4, Sox2, Klf4 ve Myc) tarafından pluripotency yeniden yapılandırılabilir ve uyarılan pluripotent kök hücreler (iPSCs) 2-4 olabildiği gösterilmiştir. HESC gibi, insan iPSCs pluripotent ve otolog hücre için potansiyel bir kaynaktır. Burada GFP içeren retroviral omurgası 4 klonlanmış dört yeniden programlama faktörleri ile insan fibroblast hücreleri yeniden programlamayı protokolü tanımlar. Aşağıdaki protokolü kullanarak, insan ESC kültür koşullarında 3-4 hafta içerisinde insan iPSCs oluşturur. İnsan iPSC koloniler yakından morfolojisi hESC benzeyen ve retroviral transgen susturulması bir sonucu olarak GFP flöresanı kaybı gösterir. iPSC koloniler bir floresan microsco altında mekanik olarak izolepe hESC gibi benzer bir şekilde davranırlar. Bu hücreler, biz çok pluripotency genlerin ve yüzey belirteçleri ifadesi algılar.

Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

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

OCT4 bir retrovirüs-aracılı ektopik ifadesi ile insan kaynaklı hücreler pluripotent kök (iPSCs) üretmek için bir yöntem olup, SOX2, KLF4 ve MYC tarif edilmektedir. GFP dayanarak insan iPSC koloniler tespit etmek pratik bir yolu da tartışılmaktadır.

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 ...

reprogramming-human-somatic-cells-into-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

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

20.4K Görüntüleme.  Yale School of Medicine. OCT4 bir retrovirüs-aracılı ektopik ifadesi ile insan kaynaklı hücreler pluripotent kök (iPSCs) üretmek için bir yöntem olup, SOX2, KLF4 ve MYC tarif edilmektedir. GFP dayanarak insan iPSC koloniler tespit etmek pratik bir yolu da tartışılmaktadır.

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

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 ...

Biyoloji

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

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

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

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

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

İnsan İsteyerek Pluripotent Kök Hücreler transfekte ve Nucleofecting

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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

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

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

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

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

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

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

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

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

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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

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

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

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

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

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

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

İnsan kardiyomiyositleri Üretimi: Besleyici-ücretsiz İnsan Bağlı Pluripotent Kök Hücre A Farklılaşma Protokolü

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...