Name: generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector
Uploaded: 2012-10-31T23:00:00
Description: Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

इन अध्ययनों के हिस्से में NIH UO1HL107443-01 जीजेएम और जीएम के लिए पुरस्कार द्वारा वित्त पोषित किया गया.

1. और परिधीय रक्त mononuclear कोशिकाओं (PBMCs) का अलगाव विस्तार 0 दिन <ol><li> एक बी.डी. Vacutainer सोडियम साइट्रेट के साथ CPT सेल तैयारी ट्यूब में परिधीय रक्त के 4 मिलीलीटर ड्रा. ट्यूब 8 से 10 बार पलटना और कमरे के तापमान पर 30 ?...

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., et al. <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+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. A., et al. <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>Takahashi, K., Tanabe, K., Ohnuki, M., et al. <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., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Stadtfeld, M., Hochedlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., et al. <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+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, H., et al. <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+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., et al. <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+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

हम यहाँ STEMCCA lentiviral वेक्टर का उपयोग करने के लिए हौसले से एकत्र परिधीय रक्त के कुछ मिलीलीटर से अलग mononuclear कोशिकाओं से मानव iPSCs उत्पन्न का वर्णन करता है. प्रोटोकॉल भी जमी PBMCs (buffy कोट से सीधे प्राप्त), महत्वपूर्ण व्या...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> BD Vacutainer सोडियम साइट्रेट के साथ CPT सेल तैयारी ट्यूब </td><td> बी.डी. बायोसाइंसेज </td><td> 362760 </td><td></td></tr><tr><td> QBSF 60 स्टेम सेल मध्यम </td><td> गुणवत्ता जैव </td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td> अटलांटा बायोलॉजिकल </td><td> S10250 </td><td></td></tr><tr><td> नॉकआउट सीरम रिप्लेसमेंट </td><td> Invitrogen </td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> चींटी बजे-2 </td><td></td></tr><tr><td> / कलम Strep </td><td> Invitrogen </td><td> 15140 </td><td></td></tr><tr><td> एल Glutamine </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> गैर आवश्यक अमीनो एसिड </td><td> Invitrogen </td><td> 11140 </td><td></td></tr><tr><td> β-mercaptoethanol </td><td> सांसद BIOMEDICALS </td><td> 190242 </td><td></td></tr><tr><td> विटामीन सी </td><td> सिग्मा </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> आर एंड डी सिस्टम </td><td> 291-G1 </td><td></td></tr><tr><td> आईएल-3 </td><td> आर एंड डी सिस्टम </td><td> 203-आईएल </td><td></td></tr><tr><td> SCF </td><td> आर एंड डी सिस्टम </td><td> 255-SC </td><td></td></tr><tr><td> ईपीओ </td><td> आर एंड डी सिस्टम </td><td> 286-EP </td><td></td></tr><tr><td> Dexamethasone </td><td> सिग्मा </td><td> D4902 </td><td></td></tr><tr><td> Polybrene </td><td> सिग्मा </td><td> H-9268 </td><td></td></tr><tr><td> bFGF </td><td> आर एंड डी सिस्टम </td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES सेल मार्कर नमूना किट </td><td> Millipore </td><td> SCR002 </td><td></td></tr></tbody></table>

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.

हम एक एकल lentiviral वेक्टर का उपयोग PBMCs से मानव iPSCs की पीढ़ी के लिए एक सरल और प्रभावी प्रोटोकॉल प्रदर्शित चित्रा 1A प्रोटोकॉल की एक योजनाबद्ध प्रतिनिधित्व दिखाता है. रक्त सोडियम साइट्रेट के साथ एक BD Vacutainer CPT ?...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector (Video) | JoVE

यहाँ हम एक एकल lentiviral reprogramming वेक्टर का उपयोग परिधीय रक्त के 3-4 मिलीग्राम से मानव iPSCs पीढ़ी के लिए एक सरल और प्रभावी प्रोटोकॉल दिखाते हैं. आसानी से उपलब्ध रक्त कोशिकाओं की Reprogramming यह एक व्यापक अनुसंधान समुदाय के लिए सुलभ बनाने के द्वारा iPSC प्रौद्योगिकी के उपयोग में तेजी लाने का वादा किया है.

मानव परिधीय रक्त से प्रेरित pluripotent स्टेम सेल का प्रयोग STEMCCA lentiviral वेक्टर पीढ़ी

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

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Biology

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

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

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

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Stemgent मानव TF lentivirus के साथ मानव Fibroblasts Reprogramming प्रेरित pluripotent स्टेम सेल की पीढ़ी सेट

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Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells (Video) | JoVE

मानव प्रेरित pluripotent स्टेम सेल Transfecting और Nucleofecting

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

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मानव प्रेरित pluripotent स्टेम सेल का चयन और अलग कालोनियों वयस्क Fibroblasts से reprogrammed किया

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