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

Эти исследования были частично финансируется за счет NIH UO1HL107443-01 Награда GJM и GM.

1. Выделение и расширение мононуклеарных клеток периферической крови (МНПК) День 0 <ol><li> Нарисуйте 4 мл периферической крови в BD Vacutainer CPT сотовых Подготовка труб с цитрат натрия. Обратить трубки 8 до 10 раз и центрифуге при 1800 мкг в течение 30 мин при комнатной температуре. В ид?...

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	<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>

Мы здесь описывать использование вектора STEMCCA лентивирусов для получения ИПСК человека из мононуклеарных клеток, выделенных из нескольких миллилитров свежесобранных периферической крови. Протокол также может быть использован для перепрограммирования замороженные РВМС (полученные ...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> BD Vacutainer CPT сотовых Подготовка труб с цитрат натрия </td><td> BD Biosciences </td><td> 362760 </td><td></td></tr><tr><td> QBSF-60 Stem Cell Средний </td><td> Качество Биологические </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> Атланта биологические </td><td> S10250 </td><td></td></tr><tr><td> Нокаут Сыворотка замена </td><td> Invitrogen </td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> Муравей-ПМ-2 </td><td></td></tr><tr><td> Pen / Strep </td><td> Invitrogen </td><td> 15140 </td><td></td></tr><tr><td> L-Глютамин </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Номера для незаменимых аминокислот </td><td> Invitrogen </td><td> 11140 </td><td></td></tr><tr><td> β-меркаптоэтанола </td><td> MP Biomedicals </td><td> 190242 </td><td></td></tr><tr><td> Аскорбиновая кислота </td><td> Сигма </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> R &amp; D Systems </td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R &amp; D Systems </td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R &amp; D Systems </td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R &amp; D Systems </td><td> 286-EP </td><td></td></tr><tr><td> Дексаметазон </td><td> Сигма </td><td> D4902 </td><td></td></tr><tr><td> Полибрен </td><td> Сигма </td><td> H-9268 </td><td></td></tr><tr><td> шФРФ </td><td> R &amp; D Systems </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 сотовых Маркер Sample Kit </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.

Мы демонстрируем простой и эффективный протокол для генерации ИПСК человека от МПК с использованием одного лентивирусов вектор. Рисунок 1А показывает схематическое представление протокола. Кровь собирали в BD Vacutainer CPT сотовых Подготовка труб с цитрат натрия, и после центрифуг...

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

Здесь мы покажем простой и эффективный протокол для генерации ИПСК человека с 3-4 мл периферической крови с помощью одной лентивирусов вектор перепрограммирования. Перепрограммирование доступны клеток крови обещает ускорить использование ИПСК технологий, сделав их доступными для более широкого научного сообщества.

Поколение человека индуцированных плюрипотентных стволовых клеток из периферической крови с помощью STEMCCA Lentiviral векторного

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

Research

JoVE Journal

Biology

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

Генерация индуцированные плюрипотентные стволовые клетки путем перепрограммирования мышиных эмбриональных фибробластов с Четыре фактора транскрипции, доксициклин индуцибельной лентивирусов системы трансдукция

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

Генерация индуцированные плюрипотентные стволовые клетки путем перепрограммирования человеческих фибробластов с Stemgent правам лентивирус TF Установить

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

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Лентивирусов-опосредованной Нокдаун В Ex Vivo Эритропоэз прав гемопоэтических стволовых клеток

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Трансфекции и Nucleofecting человека индуцированных плюрипотентных стволовых клеток

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

Выбор и изоляция Колонии человека индуцированных плюрипотентных стволовых клеток из взрослых Перепрограммированный Фибробласты

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

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

Перепрограммирование соматических клеток человека в индуцированные плюрипотентные стволовые клетки (иПСК) Использование ретровирусных вектор с GFP

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

Генерация Мыши, полученные из индуцированных плюрипотентных стволовых клеток

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Быстрая и эффективная генерация нейронов из стволовых клеток человека плюрипотентных в Multititre Формат плиты

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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

Поколению прав кардиомиоцитов: Дифференциация Протокол от Ф-свободной человеческой индуцированных плюрипотентных стволовых клеток

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

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

Эффективная генерация человека индуцированных плюрипотентных стволовых клеток из человеческих соматических клеток с Сендай-вируса

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 ...