Beschriebenen Arbeiten in diesem Manuskript wurde durch Mittel aus dem California Institute for Regenerative Medicine (CIRM) für UCR Stem Cell Core.

Unser Protokoll beginnt mit einer Methode zur menschlichen iPS zur Feeder-freien Kulturen, durch Protokolle zur Transfektion menschlichen iPS mit GeneJuice (EMD) und Nukleofektion der menschlichen iPS mit einem AMAXA nuclefector nachgeschaltet anzupassen. Hinweis: Die folgenden Verfahren werden in einem sterilen Laminarströmungshaube durchgeführt. Alle Medien und Lösungen sind äquilibriert bis 37 ° C oder Raumtemperatur bevor, wenn nicht anders angegeben. Ein. Gründung menschlichen iPS auf Feeder-free-System Menschliche iPS zuvor auf Feeder-Zellen aufrechterhalten werden aufgeteilt, übertragen auf Geltrex-beschichtete Schale und gepflegt für zwei Durchgängen vor Feeder-freie Transfektion. <ol><li> Auftauen Geltrex Nacht bei 4 ° C. Um Geltrex Beschichtung herzustellen, verdünnen aufgetaute Geltrex 1:50 in kaltem DMEM. Mischen Sie die Lösungen vorsichtig. </li></ol> Anmerkung: Geltrex wie Matrigel ist eine lösliche Form von Basalmembran matrix gereinigte aus murinen Engelbreth-Holm-Swarm (EHS) Tumorzellen. Alternativ können Matrigel als extrazelluläre Matrix Feeder-freien menschlichen iPS Kulturen zu etablieren verwendet werden. <ol start="2"><li> Die gesamte Oberfläche der Vertiefungen mit Geltrex Kultur-Lösung (1 ml für eine 35-mm-Vertiefung). Coat Vertiefungen mit Geltrex bei 37 ° C Inkubator für 1 Stunde. </li><li> Um den Durchgang menschlichen iPS, 1 ml Accutase pro Vertiefung und Inkubation bei 37 ° C für 1 min, bis die meisten Zellen zu lösen beginnen. </li></ol> Um den Durchgang menschlichen iPS, 1 ml Accutase pro Vertiefung und Inkubation bei 37 ° C für 1 min, bis die meisten Zellen zu lösen beginnen. <ol start="4"><li> Fügen 10-15 Glasperlen zu den Zellen und schwenken Sie die Platte. 2 ml KnockOut DMEM / F12 und sanft verreiben. Übertragen Zellsuspension in ein 10 ml Zentrifugenröhrchen. </li><li> Spin-Zellen bei 800 UpM für 3 min bei Raumtemperatur. </li><li> Absaugen des Überstandes aus der Röhre, so dass menschliche iPSC Pellet intakt. Gently Flick Rohrdisperse Zellpellet. </li><li> Entfernen Geltrex aus dem beschichteten gut. </li><li> Vorsichtig resuspendieren menschlichen iPS Pellet in einem geeigneten Volumen STEMPRO. Verteilen zwischen den Wells von Feedern, abhängig von der Proliferationsrate). Menschliche IPSCs kann in einem Teilungsverhältnis von 1:2 bis 1:6 passagiert werden. </li><li> Vorsichtig Ort in 5% CO 2-Inkubator, schwenken Sie die Platte vorsichtig, um eine gleichmäßige Verteilung der Zellen in den Vertiefungen zu gewährleisten. </li><li> Feed-Zellen täglich bis Zellen sind bereit, wieder aufgeteilt werden (wenn die Zellen 80% Konfluenz erreichen). </li><li> Durchgang menschlichen IPSCs auf neue Geltrex-beschichtete Vertiefung (Stufe 1.3 bis 1.9) in einem Teilungsverhältnis von 1:2. Kleine Kolonien gebildet und gleichmäßig auf Geltrex-beschichtete Vertiefung vor der Transfektion verteilt. </li></ol> 2. Transfektion von humanen iPS mit GeneJuice Zellen (Anzucht auf 6-Well-Platten) sollte ca. 40 -50% konfluenten am Tag der Transfektion optimalen Transfektionseffizienz zu erreichen. Es istnicht erforderlich, das Zellmedium bis zum nächsten Tag ändern. <ol><li> Bereiten 100 ul KO-DMEM/F12 in ein steriles 1,5 ml Eppendorf-Röhrchen. Fügen Sie 27 ul GeneJuice Transfektionsreagenz. Gut mischen. Inkubieren bei Raumtemperatur für 5 Minuten. </li><li> Add 4 pg Plasmid-DNA. Das Röhrchen bei Raumtemperatur für 15 min. Die Wahl von Plasmid ist entscheidend für eine optimale Transfektionseffizienz. Wir verwenden ein Plasmid mit einem verbesserten grüne Fluoreszenz-Protein (eGFP) durch CAG-Promotor 5 (PCAG-eGFP) angetrieben wird. CAG Promotor ein starker Promotor in humanen transkriptionell aktiven IPSCs und können daher verwendet werden, um die Expression des Transgens in diesen Zellen zu treiben ist. In unseren Händen hat Linearisierung von Plasmid nicht scheinen, um die Transfektionseffizienz beeinflussen. </li><li> Fügen GeneJuice-DNA-Gemisch zu den Zellen und schwenken Sie die Platte. Spin die Platte bei 1200 UpM für 5 Minuten (&#39;spinoculation &quot;Methode), um den Kontakt der Mischung mit Transfektion menschlicher IPSCs auf dem gut zu erhöhen. </li><li> Inkubieren der Zellen bei 37 unddeg; C über Nacht. </li><li> Monitor für Transfektionseffizienz am nächsten Tag. </li><li> Für die stabile Transfektion, ändern Medium am nächsten Tag mit frischen STEMPRO. Fügen Sie entsprechende Antibiotika-Selektion nach 24 - 48 Stunden. </li></ol> 3. Nucleofection der menschlichen iPS Menschliche iPS auf Abzweige oder Geltrex angebaut werden für Nukleofektion verwendet werden. Allerdings empfehlen wir Replattierung nucleofected menschlichen iPS auf embryonale Maus-Fibroblasten (MEF) oder menschliche Vorhaut-Fibroblast (HFF) Zubringer zu hohen Lebensfähigkeit der Zellen und Erholung zu gewährleisten. Mindestens 2 Millionen Zellen sollten verwendet werden, um höhere Zellüberleben nach Nukleofektion erreichen. <ol><li> Bereiten Feeder-Zellen konditionierten Medien (CM) durch Inkubation menschlichen iPS KnockOut Serum Replacement (KOSR) Medien mit Feeder-Zellen über Nacht. Sammle CM alle 24 Stunden. </li></ol> Anmerkung: Jeder Mausstamm zur Herstellung Feeder-Schichten (wie BLK6, CF1 und MF1) verwendet wird, ist geeignet, um daraus fo werdenr machen CM. <ol start="2"><li> Am Tag der Nukleofektion, vorzubehandeln menschlichen IPSCs mit 10 pM ROCK Inhibitors für mindestens 1 Stunde. </li><li> Bereiten Sie 82 ul des menschlichen Stammzellen Nucleofector Lösung in ein steriles 1,5 ml Eppendorf-Röhrchen. Hinzufügen 18 ul Beiblatt 1. Gut mischen. Inkubieren bei 37 ° C für 5 Minuten. </li><li> Vorwärmen Feeder-Zellen konditionierten Medien (CM) und 0,25% Trypsin / EDTA auf 37 ° C. Unter der Haube prelabel ein steriles 15 ml konischen Röhrchen. </li><li> Entfernen Sie vorsichtig Medien aus menschlichen iPS Kultur nucleofected werden. Vorsichtig waschen der Zellen mit 2 ml 1X Phosphat gepufferten Lösung (PBS) pro Vertiefung. Saugen Sie die PBS. 1 ml 0,25% Trypsin pro Vertiefung. Inkubieren Zellen bei 37 ° C für 3 Minuten. </li><li> Gently verreiben die Zellen mit 1000 ml Pipettenspitze. Waschen Sie den Boden des Bohrlochs, um sicherzustellen, dass alle menschlichen iPS vollständig abgelöst. Übertragen Zellsuspension an das markierte 15 ml konischen Röhrchen. </li></ol> Hinweis: Trypsinierung in einzelne cEllen sollte unbedingt vermieden werden. Zellen sollten nur in kleinen Klumpen von Zellen verdrängt werden bestand aus etwa Drillinge von Zellen. Kleinen Klumpen von Zellen (Zelle Tripeln) wird in einzelne Zellen dissoziiert während der nachfolgenden Handhabung der Zellen. <ol start="7"><li> 9 ml MEF Medien Trypsin zu inaktivieren. Spin-Zellen bei 800 rpm für 3 Minuten. Vorsichtig den Überstand aspirieren und lassen das Zellpellet intakt. </li><li> Die Zellen in vorgewärmten 100 ul menschlichen Stammzellen Nucleofector Lösung aus Schritt 3.3. </li><li> Übertragen Zellen einem Nucleofector Küvette unter Verwendung einer 1 ml Pipettenspitze. </li><li> Add 4 ug Plasmid-DNA in die Zellsuspension in der Küvette. Mix-Zellen und DNA durch vorsichtiges Schwenken. Tippen Sie auf die Küvette zweimal auf der Haube Oberfläche. </li><li> Legen Sie die Küvette in den Nucleofector Halter. Programme verwenden, B-016. Nucleofect Zellen durch Drücken der Taste X. </li></ol> &lt; <ok> &gt; Wird angezeigt, wenn die Nukleofektion Prozess ist complet werdened (in der Regel dauert nur 1-5 Sekunden). Die Verwendung anderer Nukleofektion Programmen (A-023, A-033 und U-023) ergab weniger als 10% eGFP-positive Zellen aus menschlichen IPSCs. </ok><ol start="12"><li> Abrufen nucleofected Zellen aus der Küvette mit dem mitgelieferten Pasteur Plastikpipette. Recover Zellen durch Resuspendieren sie in vorgewärmte CM und ROCK-Inhibitor in ein steriles 1,5 ml Eppendorf-Röhrchen. Inkubieren Zellen bei 37 ° C für 10 Minuten zu lassen Zellen erholen. </li><li> Transfer-Zellen tropfenweise auf Feeder-Schichten unter Verwendung von 1 ml Pipettenspitze. Inkubieren der Zellen bei 37 ° C über Nacht. </li><li> Monitor für Transfektionseffizienz am nächsten Tag. Zur Ableitung stabiler Klone von transgenen menschlichen iPS, dass stabil exprimieren Transgen, ändern Medium am nächsten Tag mit CM und ROCK-Inhibitor. Fügen Sie entsprechende Antibiotika-Selektion nach 24 - 48 Stunden. </li></ol> 4. Repräsentative Ergebnisse: <img alt="Abbildung 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <strong&gt; Abbildung 1. Mikrophotographien Riv1 menschlichen iPS mit PCAG-eGFP transfiziert. (A) eGFP-exprimierenden Zellen transfiziert mit Riv1 GeneJuice auf Geltrex 12 Stunden nach der Transfektion. Kolonien von Riv1 menschlichen IPSCs ausplattiert und gebildet auf Zubringer nach Nukleofektion mit B-016 (B) und A-023 (C)-Programm. (D) Stabil eGFP-exprimierenden humanen iPS Kolonien mit allgegenwärtigen eGFP Expression von GeneJuice abgeleitet. (E) Stabil transfizierte Riv1 menschlichen iPS erhalten konstitutive eGFP Expression während Embryoidkörper Differenzierung.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., 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=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

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Unsere Protokolle führen einfache, robuste und hoch reproduzierbare Techniken, um Transgene in menschlichen iPS einzuführen, ohne prominente toxische Wirkung und Zelltod. Menschliche IPSCs sollte in kleinere Klumpen von Zellen (5-10 Zellen) passagiert und plattiert auf Geltrex bei hoher Dichte (1:2), um eine optimale Effizienz der Transfektion in zahlreichen kleinen Kolonien gewährleisten. Für menschliche iPSC Linien, die anfälliger für Differenzierung und Zelltod sind, sollten höhere Zahl menschlicher IPSCs (bis 4 x 10 6 Zellen) für eine einzelne Nukleofektion Experiment verwendet werden. Transienten Transfektions-Assay erzeugt eine große Anzahl von Transgen-exprimierenden humanen IPSCs innerhalb von 1 Tag. Stabil transfizierte iPSC Klone treten in der Regel innerhalb von 7 Tagen, und diese transgenen Kolonien sollten bereit sein, innerhalb von drei Wochen abgeholt werden. Die Verwendung von CAG-Promotor hier beschriebenen sorgt ubiquitäre Expression von eGFP Reporter. Unter solchen Verbesserungen können unsere Protokolle in anderen Anwendungen zugeführt werden, einschließlich Überexpression, conditional Induktion Ableitung linienspezifischen Reporter Linien, shRNA oder siRNA knockdown, Gen-Targeting und homologe Rekombination.

<table border="1"><tbody><tr><td> Namen Reagenz </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 0,25% Trypsin mit EDTA </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2-Mercaptoethanol </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3 mm Glaskugeln </td><td> Fisher Scientific </td><td> 11-312A </td><td> Glaskügelchen sollte mit Salzsäure (HCl) über Nacht, abgerundet mit Natriumhydroxid (NaOH) und destilliertem Wasser gespült werden und sterilisiert durch Autoklav vor der Verwendung. </td></tr><tr><td> Accutase Zelldissoziationslösung Reagenz </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> Basischen Fibroblastenwachstumsfaktor (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (high glucose) </td&gt; <td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> Fötales Kälberserum </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> Growth Faktor reduziert </td></tr><tr><td> GeneJuice Transfektionsreagenz </td><td> EMD Biosciences </td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I (100X) </td><td> Invitrogen </td><td> 35050061 </td><td> L-Glutamin (Invitrogen, 25.030.081) kann auch stattdessen verwendet werden. </td></tr><tr><td> Menschliche iPS KOSR Medien </td><td></td><td></td><td> 500 ml Medium menschlichen ES besteht aus 390 ml KnockOut DMEM / F12, 100 ml Serum KnockOut Replacer, 5ml Glutamax-I (100X), 5ml NEAA (100X), 500 ul 2-Mercaptoethanol (55mm) und mit 10 ng / ml bFGF . </td></tr><tr><td> KnockOut DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td>KnockOut Serum Replacement (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Maus embryonale Fibroblasten Medien </td><td></td><td></td><td> MEF Medien besteht aus 90% FBS, 1X NEAA, 1X Glutamax-I und 1X Natriumpyruvat in DMEM (hoher Glucose) </td></tr><tr><td> Nicht essentielle Aminosäure, NEAA? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> Menschliche Stammzellen Nucleofector Lösung 1 mit Zuschlag </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> Phosphate Buffered ohne Ca 2 Saline + und Mg 2 + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> Natriumpyruvat (100X) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO Medium Kit </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ml STEMPRO media besteht aus 454 ml KlopfOut DMEM / F12, 10 ml STEMPRO serumfreies Wachstum Supplement (50X), 5ml Glutamax-I (100X), 5ml NEAA (100X), 36 ml BSA (Bovine Serum Albumin, 25%), 909 ul 2-Mercaptoethanol (55mm ) und versetzt mit 10 ng / ml bFGF. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

Genetische Veränderung ist weiterhin ein wichtiges Instrument sein, das Studium der Stammzellbiologie und darlegt mögliche klinische Anwendungen von humanen embryonalen Stammzellen (hES-Zellen) 1. Während Verbesserungen in mehreren Gentransfer Methoden 2-9 beschrieben haben, bleibt Transfektion ein launischer Verfahren zur hESCs, und noch nicht in der menschlichen induzierten pluripotenten Stammzellen (iPS) berichtet worden. In diesem Video zeigen wir, wie unser Labor routinemäßig transfiziert und nucleofects menschlichen iPS Verwendung mit einem erweiterten grün fluoreszierendes Protein (eGFP) Reporterplasmid. Menschliche IPSCs angepasst und aufrechterhalten, wie Feeder-freien Kulturen, die Möglichkeit der Zuführung Zelltransfektion beseitigen und um eine effiziente Selektion stabiler transgener iPSC Klone nach der Transfektion ermöglichen. Für Nukleofektion werden menschliche iPS mit ROCK-Inhibitor 11, in kleine Klumpen von Zellen, nucleofected und Zubringer replattiert in Feederzellschicht klimatisierten trypsiniert vorbehandeltmittel-bis Zellgewinnung verbessern. Transgen-exprimierenden menschlichen iPS kann nach 6 Stunden erreicht werden. Antibiotika-Selektion wird nach 24 Stunden und stabilen transgenen Linien erscheinen innerhalb 1 Woche angewendet. Unser Protokoll ist robust und reproduzierbar für menschliche iPSC Leitungen ohne Änderung Pluripotenz dieser Zellen.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Trotz der jüngsten Fortschritte in der Gentechnik, bleibt Transfektion von humanen embryonalen Stammzellen (hES) ein launischer Prozess. Nach unserem Kenntnisstand haben systematische und effiziente Methoden, um Menschen induzierte pluripotente Stammzellen (iPS) transfizieren nicht berichtet worden. Hier beschreiben wir robuste Protokolle effizient transfizieren und nucleofect menschlichen iPS.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.3K Aufrufe.  University of California Riverside. Trotz der jüngsten Fortschritte in der Gentechnik, bleibt Transfektion von humanen embryonalen Stammzellen (hES) ein launischer Prozess. Nach unserem Kenntnisstand haben systematische und effiziente Methoden, um Menschen induzierte pluripotente Stammzellen (iPS) transfizieren nicht berichtet worden. Hier beschreiben wir robuste Protokolle effizient transfizieren und nucleofect menschlichen iPS.

Serie: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfektion humaner neuraler Stammzellen mit dem Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Forschung

Biologie

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.

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 ...

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 ...

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.

Vorbereitung von embryonalen Maus-Fibroblasten-Zellen zum Kultivieren von humanen embryonalen und induzierten pluripotenten Stammzellen

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.

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.