1. Reprogrammierung <ol><li> Seed BJ-Zellen bei einer Dichte von 1 x 10 5 Zellen pro Vertiefung einer 6-Well-Platte. Kultur die Zellen in 2 ml Wachstumsmedium (450 ml EMEM mit 50 ml ES qualifizierten FBS, 5 ml 10 mM nicht-essentiellen Aminosäuren, 5 ml Penicillin / Streptomycin, 5 ml 200 mM L-Glutamin und 0,9 ml 55 mM β ergänzt -Mercaptoethanol) über Nacht bei 37 ° C und 5% CO 2. </li><li> Am selben Tag, beginnt die Vorbereitung MEF Feeder-Platten durch Zugabe von 0,1% Gelatine in Wasser zu einer 6-Well Platte verdünnt und Inkubation über Nacht bei 37 ° C und 5% CO 2. </li><li> Nach der Inkubation zu entfernen Medium aus dem BJ-Zellen und 2 ml Wachstumsmedium mit 6 pg / ml Polybren ergänzt, 500 ul hOct4-Lentivirus, 50 ul hSox2-Lentivirus, 50 ul hNanog-Lentivirus und 50 ul hLin28-Lentivirus in jede Vertiefung . Schütteln Sie die Platte, um eine gleichmäßige Beschichtung des Mediums zu gewährleisten. Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Am selben Tag, entfernen Sie 1 Flasche CF-1 MEF-Zellen aus flüssigem Stickstoff und Auftauen. Entfernen von Flüssigkeit aus der Gelatine beschichteten Vertiefungen (siehe Schritt 1.2) und fügen MEF Feeder-Zellen bei einer Dichte von 0,2 x 10 5 Zellen / well. Das Gesamtvolumen pro Vertiefung sollte 2ml Zellen in MEF Wachstumsmedium (450 ml DMEM, ergänzt mit 50 ml FBS und 5 ml nicht-essentielle Aminosäuren). </li><li> Am nächsten Tag lösen BJ-Zellen mit 0,05% Trypsin / EDTA und Zentrifuge bei 200 xg für 5 Minuten. Saugen Sie das Medium und Zellen in Wachstumsmedium. Entfernen Sie Medium von der MEF Feeder-Platte (siehe Schritt 1,4) und 2 ml / well des BJ Zellsuspension. Die Konzentration der Zellen sollte ca. 5 x 10 4 Zellen / well werden. Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Ersetzen Medium 24 Stunden nach der erneuten Aussaat mit menschlichen ES / iPS Zellkulturmedium (400 ml DMEM/F12 mit 100 ml Knockout Serum Replacement, 5 ml 10 mM nicht-essentiellen Aminosäuren, 5 ml 200 mM L-Glutamin, 0,9 ml ergänzt 55 mM β-Mercaptoethanol und 20 ng / ml humanes rekombinantes bFGF). Ändern Medium alle 24 Stunden für 7 Tage. </li><li> Nach sieben Tagen ändern mittel-bis MEF konditionierten Medium (siehe Abschnitt 2). </li></ol> 2. Vorbereitung der MEF konditioniertem Medium <ol><li> Seed CF-1 MEF Feeder-Zellen bei 2 x 10 5 Zellen / well auf einer 6-Well-Platte in 2 ml MEF Wachstumsmedium. Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Ändern Medium für die menschliche ES / iPS Zellkulturmedium. Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Sammeln Überstand alle 24 Stunden für vier Tage. Den Überstand durch ein 0,22 um-Filter. </li><li> Supplement Überstand mit 50 ng / ml bFGF. </li></ol> 3. iPS Colony Auswahl und Passagierung <ol><li> Wählen Sie ein ES-ähnliche Kolonie und re-Samen in menschlichen ES / iPS Kulturmedium mit 10 uM Stemolecule Y27632 auf Zellkulturschalen mit CF-1 MEF Feeder-Zellen vor ausgesät ergänzt. Inkubieren über Nacht bei 37 ° C und 5% CO 2. </li><li> Ändern Sie das Kulturmedium alle 24 Stunden für die ersten 7 Tage (ohne Ergänzung mit Stemolecule Y27632). Nach sieben Tagen ändern mittel-bis MEF konditionierten Medium (siehe Abschnitt 2 für die Vorbereitung). Wir setzten die Passage der Zellen, bis sie typischen menschlichen ES Morphologie zeigten. </li></ol> 4. Immunzytochemische Untersuchung der Pluripotenz Markers <ol><li> Waschen Sie die Zellen vorsichtig dreimal mit PBS. </li><li> Fix die Zellen mit 500 ul Fixativ für 20 Minuten bei Raumtemperatur. </li><li> Waschen Sie die Zellen vorsichtig dreimal mit PBS. </li><li> Block unspezifische Bindung mit 500 ul Blockierungspuffer für eine Stunde bei Raumtemperatur. </li><li> Inkubieren Sie die Zellen mit 250 ul der spezifischen Primärantikörper über Nacht bei 4 ° C (wir TRA-1 bis 81, TRA-1 bis 60, SSEA-4, SECO-3, Oct4, Sox2, Nanog und Lin28 bei 1:100 Verdünnungen). </li><li> Waschen Sie die Zellen vorsichtig dreimal mit PBS. </li><li> Inkubieren Sie die Zellen mit 250 ul des sekundären Antikörper für 1 Stunde bei Raumtemperatur, hielten sich fern von Licht (wir Goat anti-Mouse IgM Cy3-Konjugat, Ziege anti-Maus IgG Cy3-Konjugat, Ziege anti-Ratte IgM Cy3-Konjugat und Ziege anti- Kaninchen Cy3-Konjugat bei 1:300 Verdünnungen). </li><li> Waschen Sie die Zellen vorsichtig dreimal mit PBS. </li><li> Add DAPI (Endkonzentration 1 pg / ml) auf dem letzten Waschen und Inkubation 5 Minuten, um Kerne zu visualisieren. </li><li> Analysieren Sie die Zellen unter Vergrößerung. </li></ol> Teil 5: Repräsentative Ergebnisse 1. Morphologie Ergebnisse: Menschliche Fibroblasten Vorhaut (BJ)-Zellen wurden co-transduzierten mit Oct4, Sox2, Nanog und Lin28. Morphologische Veränderungen wurden so früh wie Tag 4 nach der Transduktion beobachtet, und die Ansammlung von Zellen wurde stärker an Tag 17 (Abbildung 1) verpackt. Kolonien wurden manuell am Tag 25 aufgenommen und kultiviert auf CF-1 MEF Feeder-Zellen. Zur Erleichterung der iPS-Zelle Koloniebildung nach der Neuprogrammierung verwendeten wir Stemolecule Y27632, Eine ROCK-Inhibitor, für die erste Nacht Aussaat bei jedem Durchgang. iPS Zellkolonien mit guter Morphologie wurden nach drei aufeinanderfolgenden Runden Kolonie Kommissionierung und Passagieren beobachtet. 2. Expression der Pluripotenz Marker: Zur weiteren Charakterisierung der isolierten iPS Zellkolonien, schauten wir auf das Vorhandensein von gemeinsamen Pluripotenz Marker in ES-Zellen exprimiert. Die Kolonien zeigten starke alkalische Phosphatase (AP)-Aktivität (Abbildung 2). Darüber hinaus wurde Immunzytochemie (ICC) auf die iPS-Zellen Kolonien mit einem Panel von Pluripotenz-Marker-spezifischen Antikörper durchgeführt, darunter Oberflächenmarker TRA-1 bis 81, TRA-1 bis 60, SECO-4 und SSEA-3 sowie nukleare Marker Oct4, Sox2 und Nanog. Die isolierte iPS Kolonien wurden positiv für alle Marker (Abbildung 3). Die ICC Ergebnisse zeigen, dass die iPS-Zellen die entsprechende Pluripotenz-Marker Expressionsmuster ausgestellt, die zeigen, dass diese iPS-Zellen ähneln undifferenzierten humanen ES-Zellen. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Morphologische Veränderungen der transduzierten BJ-Zellen. Hellfeld Bilder von einem typischen iPS-Zelle Kolonie (A) 4 Tage und (B) 17 Tage nach der Transduktion gebildet. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Abbildung 2" /> Abbildung 2: AP-Aktivität von umprogrammiert BJ-Zellen. Drei verschiedene Kolonien mit Stemgent alkalische Phosphatase-Färbung Kit gefärbt. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Human iPS-Zellen exprimieren ein hohes Maß an folgende ES-Zell-spezifische Marker: Oberflächenmarker TRA-1 bis 81, TRA-1 bis 60, SECO-4 und SSEA-3, und nukleare Marker 4 Oktober, Nanog und Sox2.

<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>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

Diese Ergebnisse zeigen, dass die Stemgent Menschliche TF Lentivirus Set verwendet werden, um effizient zu generieren iPS Kolonien durch Induktion der ektopische Expression von transduziert werden Transkriptionsfaktoren in humanen Fibroblasten. Bei der Gestaltung Umprogrammierung Experimente sollten mehrere Variablen berücksichtigt, um die Effizienz der Reprogrammierung zu optimieren. Erstens kann der aktive Virus-to-Target-Verhältnis (Multiplizität der Infektion MOI) müssen während der primären Transduktion Schritt, um eine optimale Effizienz zu erreichen Transduktion modifiziert werden. Zweitens kann das Wachstum Zustand der Zielzellen Auswirkungen Neuprogrammierung. Gesunde und vermehrungsfähige Zellen sind leichter zu Neuprogrammierung. Drittens, wenn Änderung des Protokolls für verschiedene Zell-Zahlen, ist es empfehlenswert, Zielzelle Zahlen proportional zur Oberfläche der Kulturschale eingestellt werden. Schließlich sollte die Anwendung ROCK-Inhibitoren wie Y27632 betrachtet, um sicherzustellen, erfolgreiche Reprogrammierung wie jüngste Studien ihren Nutzen bei der Verbesserung der hES Kolonie Überleben nachweisbar sind. 4,5

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<td>Human TF Lentivirus Set</td>
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<td>TRA-1-81 antibody</td>
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<td>&nbsp;</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

Im Jahr 2006 Yamanaka und seine Kollegen zunächst nach, dass Retrovirus-vermittelte Lieferung und die Expression von Oct4, Sox2, c-Myc und Klf4 induzieren können die pluripotenten Zustand in Maus-Fibroblasten ist. 1 Die gleiche Gruppe berichtete auch die erfolgreiche Reprogrammierung von menschlichen Körperzellen in induzierte pluripotente Stammzellen (iPS-Zellen) unter Verwendung von humanen Versionen des gleichen Transkriptionsfaktoren, die durch retrovirale Vektoren geliefert. 2 Zusätzlich berichtete James Thomson et al., dass die Lentivirus-vermittelte Co-Expression von einem anderen Satz von Faktoren (Oct4, Sox2, Nanog und Lin28 ) war in der Lage Reprogrammierung menschlicher Körperzellen in iPS-Zellen. 3 iPS-Zellen ähneln embryonalen Stammzellen in der Morphologie, Proliferation und die Fähigkeit, sich in alle Gewebetypen des Körpers differenzieren. Menschliche iPS-Zellen haben einen entscheidenden Vorteil gegenüber embryonalen Stammzellen, da sie wichtige Eigenschaften von ES-Zellen ohne das ethische Dilemma der Embryo zerstört aufweisen. Die Erzeugung von Patienten-spezifischen iPS-Zellen umgeht eine wichtige Hürde, um persönliche regenerativen Medizin Therapien durch den Wegfall der Potenzial für Immunabwehr von nicht-autologen transplantierten Zellen. Hier zeigen wir das Protokoll für die Reprogrammierung menschlicher Fibroblasten-Zellen unter Verwendung des Stemgent Menschliche TF Lentivirus Set. Wir zeigen auch, dass Zellen mit diesem Satz neu programmiert, um iPS zeigen Morphologie vier Tage nach der Transduktion beginnen. Mit dem Stemolecule Y27632, wählten wir für iPS-Zellen und beobachtet richtige Morphologie nach drei aufeinanderfolgenden Runden Kolonie Kommissionierung und Passagieren. Wir zeigen auch, dass nach der Neuprogrammierung Zellen der Pluripotenz-Marker AP, Oberflächenmarker TRA-1 bis 81, TRA-1 bis 60, SECO-4 und SSEA-3, und nukleare Marker Oct4, Sox2 und Nanog angezeigt.

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

Wir zeigen das Protokoll für die Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Lentivirus-vermittelte Lieferung der menschlichen Faktoren Oct4, Sox2, Nanog und Lin28. Pluripotenz von Morphologie und die Anwesenheit von embryonalen Stammzellen (ES) Zell-spezifische Marker bestätigt.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung menschlichen Fibroblasten mit dem Stemgent Menschliche 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 ...

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Embryonic and Induced Pluripotent Stem Cells

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29.7K Aufrufe.  Stemgent. Wir zeigen das Protokoll für die Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Lentivirus-vermittelte Lieferung der menschlichen Faktoren Oct4, Sox2, Nanog und Lin28. Pluripotenz von Morphologie und die Anwesenheit von embryonalen Stammzellen (ES) Zell-spezifische Marker bestätigt.

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

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.

Kultur und Wartung von humanen embryonalen Stammzellen

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

Forschung

Biologie

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.

Erzeugung von induzierten pluripotenten Stammzellen durch Reprogrammierung embryonalen Maus-Fibroblasten mit einem Vier Transcription Factor, induzierbare Doxycycline Lentivirale Transduktionssystems

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.

Einfrieren und Auftauen humanen embryonalen Stammzellen

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 ...

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.

Transfektion und Nucleofecting menschlichen induzierten pluripotenten Stammzellen

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.

Selektieren und Isolieren Colonies of Human induzierten pluripotenten Stammzellen aus adulten Fibroblasten Umprogrammierte

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.

Reprogrammierung menschlichen Körperzellen in induzierte pluripotente Stammzellen (IPSCs) mit retroviralen Vektoren mit 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

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.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

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.

Generation of Human induzierten pluripotenten Stammzellen aus dem peripheren Blut mit dem STEMCCA Lentivirusvektor

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.

Generation of Human Cardiomyocytes: Eine Differenzierung Protokoll von Feeder-freien menschlichen induzierten pluripotenten Stammzellen

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

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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

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 ...