Name: reprogramming human somatic cells into induced pluripotent stem cells (ipscs) using retroviral vector with gfp
Uploaded: 2012-04-03T12:00:00
Description: Watch this Scientific Journal Video about Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP at JoVE.com

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 ...

<ol>
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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Hotta, A., Ellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+vector+silencing+during+iPS+cell+induction:+an+epigenetic+beacon+that+signals+distinct+pluripotent+states.">Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states.</a> Journal of Cellular Biochemistry. 105, 940-948 (2008).</li><li>Matsui, T., Leung, D., Miyashita, H., Maksakova, I. A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M. C., Shinkai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proviral+silencing+in+embryonic+stem+cells+requires+the+histone+methyltransferase+ESET.">Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET.</a> Nature. 464, 927-931 (2010).</li><li>Wolf, D., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+use+ZFP809+to+silence+retroviral+DNAs.">Embryonic stem cells use ZFP809 to silence retroviral DNAs.</a> Nature. 458, 1201-1204 (2009).</li><li>Chan, E. M., Ratanasirintrawoot, S., Park, I. H., Manos, P. D., Loh, Y. H., Huo, H., Miller, J. D., Hartung, O., Rho, J., Ince, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+distinguishes+bona+fide+human+iPS+cells+from+partially+reprogrammed+cells.">Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.</a> Nat. Biotechnol. 27, 1033-1037 (2009).</li><li>Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, ., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R. <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+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., Ebina, W., Mandal, P. K., Smith, Z. D., Meissner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li><li>Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+transgene-free+human+induced+pluripotent+stem+cells+(iPSCs)+by+temperature-sensitive+Sendai+virus+vectors.">Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 14234-14239 (2011).</li><li>Wolf, D., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRIM28+mediates+primer+binding+site-targeted+silencing+of+murine+leukemia+virus+in+embryonic+cells.">TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells.</a> Cell. 131, 46-57 (2007).</li><li>Park, I. H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M. W., Cowan, C., Hochedlinger, K., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Kim, K. Y., Hysolli, E., 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=Neuronal+maturation+defect+in+induced+pluripotent+stem+cells+from+patients+with+Rett+syndrome.">Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 14169-14174 (2011).</li></ol>

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 ...

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Biology

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

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 ...

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

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

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

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 ...

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

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

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

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

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 ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...