Wir möchten den Mitgliedern des Lee-Labor für wertvolle Diskussionen zum Manuskript danken. Die Arbeit im Labor Lee wurde durch Zuschüsse von Robertson Investigator Award der New York Stem Cell Foundation und von Maryland Stem Cell Research Fund (TEDCO) unterstützt.

1. Herstellung von Zell und Medien (Tag 1) <ol><li> Kultur und erweitern menschlichen Fibroblasten mit DMEM-Medium mit 10% FBS. </li><li> Teller menschlichen Fibroblasten (Abbildung 1) auf eine 24-Well-Platte auf der entsprechenden Dichte pro Well am Tag vor der Transduktion. HINWEIS: Die folgenden serielle Verdünnungen werden empfohlen (200K, 100K, 50K, 25K, 12.5K und 6.25K), weil verschiedene Zelltypen haben unterschiedliche Befestigungs Fähigkeit. </li><li> Die Zellen für einen Tag Inkubation in einem 37 ° C, 5% CO 2-Inkubator, wodurch die Zellen vollflächig verklebt und erweitert. </li></ol> 2. Führen Transduction (Tag 2) <ol><li> Am Tag der Transduktion, überprüfen Sie die Zelldichte und wählte die effizientesten Dichte Brunnen (Abbildung 2). Es ist besser, drei verschiedenen Dichten wählen: hoch (80 ~ 90%), Mitte (50 ~ 70%) und niedriger (20 ~ 40%). </li><li> Mindestens 1 Stunde vor Übertragungs absaugen fibroblast Medien aus den Zellen und verändern neue 300 ul von Fibroblasten-Medium. </li><li> Entfernen Sie einen Satz von 4 verschiedenen Sendai-Virus-Rohre von -80 ° C-Lagerung. Auftauen jedes Rohr in der gleichen Zeit in einem 37 ° C Wasserbad für einige Sekunden und dann die Rohre aus dem Wasserbad. Danach auftauen sie auf Raumtemperatur; Zentrifugenröhrchen bei 6000 g für 10 sec und legen sie auf Eis, bis Gebrauch. Wieder einfrieren und auftauen Sie nicht das Virus, da die Titer nicht aufrecht zu erhalten. </li><li> Hinzufügen der angegebenen Mengen von jedem der vier Röhrchen Sendai-Virus (Oct-4, Klf-4, c-Myc und Sox-2) auf den Mikrozentrifugenröhrchen. Stellen Sie sicher, dass die Lösung auch durch Pipettieren vorsichtig gemischt. Zum Beispiel, wenn 50K Zellen / ein gut 24-Well-Platte für die Transduktion Lookwell: (Titer auf der Grundlage der Analysezertifikat von Life Technologies, kann von Charge zu Charge sein) Rohr hOct-4 = 6,0 x 10 7 CIU, 3 MOI = 5 ul Rohr hSox-2 = 6,5 x 10 7 CIU, 3 MOI = 4,6 μ; L Rohr hKlf-4 = 6,3 x 10 7 CIU, 3 MOI = 4,8 ul U-hc-Myc = 7,8 x 10 7 CIU, 3 MOI = 3,8 ul Total = 18,2 ul von vier Faktoren Virus Mischung / ein gut (50K-Zellen) von 24-Well-Platte </li><li> Je nach Zelldichte; hinzufügen 2X, 1X, 0,5 X und Volumen der Virusgemisch in den Brunnen. Schütteln Sie die Platte von vorne nach hinten, links und rechts (2X: 36,4 ul Virus Mischung, 1X: 18,2 ul 0,5 X und 9,1 ul). </li><li> Platzieren der Zellen in ein 37 º C, 5% CO 2-Inkubator über Nacht inkubieren. </li></ol> 3. Austausch von Kultur-Medium (Tag 3 und Tag 5) <ol><li> 24 Stunden nach der Transduktion, ersetzen Sie das Medium mit 500 ul frisch Fibroblasten-Medium. </li><li> An Tag 5, verändern Medium mit frischen Fibroblasten-Medien. </li></ol> 4. Bereiten MEF Geschirr für die Co-Kultur (Tag 8) <ol><li> Bereiten MEF-Zellen in 60 mm-Kulturschalen mit 10% FBS enthielt DMEM-Medium, da einy vor Passagieren des transduzierten Fibroblasten auf MEF Feeder-Zellen. Hinweis: Wir sind mit 5 x 10 5 von MEF in jedem 60 mm-Schale. </li></ol> 5. Starten Co-Kultur Transduzierte MEF-Zellen mit Feeder-Zellen (Tag 9) <ol><li> Vor der Beschichtung transduzierten Zellen absaugen 10% FBS enthielt DMEM-Medium von MEF Feeder-Gerichte und fügen Sie frisches 10% FBS DMEM-Medium enthalten ist. </li><li> Entfernen des Mediums von den transduzierten Fibroblasten, die in der 24-Well-Platte und wasche die Zellen einmal mit D-PBS. Nach Zugabe von 200 ul 0,25% Trypsin / EDTA in die Fibroblastenzellen inder 24-Well-Platte. Nach weniger als 5 min Inkubation mit Trypsin / EDTA, wenn die Zellen beginnen, von der Platte zu lösen, sammeln Sie die Zellen mit Wachstumsmedium in 15 ml konischen Röhrchen. Dann zentrifugieren in 6000 g für 4 min. HINWEIS: Die Bündelung 2X, 1X und 0,5 X Virus transduzierten Fibroblasten wird empfohlen. </li><li> Entfernen Sie das überstehende Medium und zu waschen, um erneut zu suspendierendas Zellpellet mit 4-5 ml frischem Fibroblasten-Medium für die Neutralisation von Trypsin. Dann zentrifugieren bei 135 × g für 4 min. </li><li> Platte Zellen als serielle Verdünnung Dichte und beginnen zu Kokultur mit Fibroblasten-Medium auf MEF: z. B. 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 und 1/128 pro 60 mm-Schale. Je nach Zellsterberate während der ersten Woche der Transduktion kann man nicht zu auf 1/2 oder 1/128 Dichte zu verdünnen. Beiseite verbleibenden Zellen Gesamt-RNA als Positivkontrolle für Transgene RT-PCR zu extrahieren. </li><li> Platz 60 mm Schalen in 37 ° C, 5% CO 2-Inkubator über Nacht. </li></ol> 6. Feed-menschlichen embryonalen Stammzellen Medium und Überwachung der Zellen (Tag 10) <ol><li> Am nächsten Tag ändern Fibroblasten-Medien auf menschliche ES-Medien mit 10 uM Y-27632. Danach ändern Medium täglich mit menschlichen ES-Medium: 800 ml + 200 ml DMEM/F12 Knockout Serumersatz + 10 ml L-Glutamin + 10 ml 10 mM MEM Mindest nicht-essentiellen Aminosäuren+ 1 ml 2-Mercaptoethanol + 10 ng / ml basischen Fibroblastenwachstumsfaktor. </li><li> Führen Sie eine tägliche Medienwechsel mit frischen menschlichen ES-Medien. Nach einer Woche, prüfen die Gerichte einmal pro 2 ~ 3 Tage unter einem Mikroskop für die Bildung von ES-Kolonie-wie Zellklumpen. Je nach Zelltyp, wird die Kultur mehr oder weniger Zeit in Anspruch nehmen, bevor Kolonien gefunden. </li></ol> 7. Picking die induzierten pluripotenten Stammzellen Kolonien und erweitern die Zellen (Tag 20 ~) <ol><li> Drei Wochen nach Transduktion, sollte Kolonien richtig für die Kommissionierung gewachsen erscheinen. </li><li> Am Tag vor der Kommissionierung die Kolonien, bereiten MEF Feeder im 24-Well-Platte. HINWEIS: Wir sind mit 12,5 x 10 5 von MEF in einer 24-Well-Platte. </li><li> Am nächsten Tag ändern Medium der MEF Gerichte aus Fibroblasten-Medien auf menschliche ES-Medien mit 10 uM Y-27632. Manuell holen eine Kolonie zu jeder Zeit unter dem Mikroskop in der Kommissionierung-Haube und machen kleinere Klumpen durch Pipettieren und übertragen Sie sieauf neu erstellten MEF dishes.Try mehrere Klone holen. </li><li> Passage und erweitern die jeweils auch von einer 24-Well-Platte mit einer 6-Well-Platte zunächst. Dann aus einer 6-Well-Platte auf eine 60-mm-Schalen, bevor die weitere Vermehrung. </li></ol> 8. Charakterisierung humaner iPS-Zellen (nach 10 Passagen) <ol><li> Immunfluoreszenz <ol><li> Teller menschlichen iPS-Zellen in 24-Well-Platte und Kultur während 4-5 Tage mit täglichen Medienwechsel. </li><li> Zum Starten Immunfluoreszenz-Test, waschen Sie die Platte mit D-PBS einmal. Dann fixieren Sie die Zellen sofort in 0,4% Paraformaldehyd für 30 min. Danach waschen dreimal in D-PBS für 5 min. </li><li> Behandlung von fixierten Zellen mit 0,1% Triton X-100-Lösung in PBS für 15 min bei Raumtemperatur. </li><li> Absaugen Permeabilisierung Lösung (0,1% Triton X-100-Lösung), dann mit 0,5% BSA-Blockierungslösung für 1 Stunde bei Raumtemperatur. </li><li> Führen Sie eine Erkennung von Nanog, Oct-4, SSEA (Stufe spezifischen embryonalen Antigde) und TRA-1 bis 81 (Tumorantigen) mit einem Anti-Human-Antikörper in 0,5% BSA-Lösung (überprüfen Sie die Materialtabelle für die Antikörperverdünnungsrate). Inkubieren des primären Antikörpers für 2 h bei Raumtemperatur. Dann waschen dreimal in D-PBS für jeweils 5 min. </li><li> Überprüfen mittels Fluoreszenzmikroskopie nach 1 h Inkubation mit Alexa Fluor 488 Ziege anti-Maus-IgG-Antikörper als 2. Antikörper, der 1:2000 in 0,5% BSA-Lösung bei Raumtemperatur verdünnt. Stain alle Kerne von je MEF Feeder-Zellen und hiPSCs mit DAPI. </li></ol></li><li> RT-PCR von Transgene Bestätigung <ol><li> Auszug Gesamt-mRNA aus Zellpellet durch RNA-Extraktion mit Reagenzien. </li><li> Ein Reverse Transkription von Aliquoten (1 ug) von Gesamt-RNA und die resultierende cDNA für die PCR-Amplifikation durch reverse Transkriptase. </li><li> Die PCR-Gemische zu 2 ul cDNA-Matrize wurden 10 ul 2X PCR-Master-Mix, 2 &amp;mgr; l von 2 pmol Primer enthalten(Vorwärts: 1 ul-und Rückwärts: 1 ul) und 6 ul der DW. Um die Genexpression zu erfassen, führen die RT-PCR mit den in Tabelle 1 angegebenen Primer. </li><li> Führen die RT-PCR mit GAPDH-Gen als Kontrolle für die Effizienz der Amplifikationsreaktionen. Visualisieren und PCR-Produkt analysieren, um 1% Agarose-Gel-Elektrophorese. </li></ol></li></ol>

<ol>
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Die Autoren haben nichts zu offenbaren.

Reprogrammierung menschlicher Körperzellen zu hiPSCs hält beispiellosen Versprechungen in Grundlagen der Biologie, persönliche Medizin, Transplantation und 12. Zuvor benötigte menschlichen iPS Generationen DNA-Virus, das Integrationsrisiko in das Wirtsgenom, die unerwünschte genetische Mutationen, die weitere klinische Anwendungen wie Arzneimittelentwicklung und Transplantationstherapien 13 begrenzen erstellen hat. Mit aus diesem Grund haben viele Studien wurde berichtet, Vektor-und gentechnik...

Menschliche embryonale Stammzellen (hES-Zellen) haben eine Kapazität zur Selbsterneuerung in vitro und haben Pluripotenz, die potenziell nützlich für Krankheit Modellierung sein könnte, für Wirkstoff-Screening, und zellbasierte Therapien zu entwickeln, um Krankheiten und Gewebeverletzungen zu behandeln. Allerdings haben hESCs eine Beschränkung für die Zell-Ersatztherapie wegen der immunologischen, onkologischen und ethischen Schranken, und im Zusammenhang mit Krankheit Gene studieren, konnte krankheitsspezifische hESCs durch Präimplantations-Diagnostik (PID) Ansätze isoliert werden, aber es ist immer noch eine technische Herausforderung und die Embryo-Spenden sind ziemlich selten. Diese Fragen werden an die Fortschritte in der Stammzellbiologie, die zur Entwicklung von induzierten pluripotenten Stammzellen (hiPSCs) geführt hat, zusammen. Menschliche iPS sind genetisch aus menschlichen adulten somatischen Zellen umprogrammiert und Hafen pluripotente Stammzelle-ähnliche Funktionen ähnlich hESCs, der sie macht eine nützliche Quelle für die regenerative Medizin wie dTeppich Entdeckung, Krankheit Modellierung und Zelltherapie in Patienten-spezifischen Weise 1,2. Bis jetzt gibt es mehrere Methoden, um menschliche iPS-Zellen zu generieren, einschließlich Virus-vermittelte (Retrovirus und Adenovirus) 3, nicht-Virus-vermittelte (BAC-System und Vektoren Transfektion) 4-Gen Transduktionen und Protein-Liefersystem 7.5. Obwohl eine Lieferung von Virus-vermittelte Gene können ein gewisses Maß an Effizienz zu gewährleisten, konnte virale Vektoren genetischen Fußabdruck zu verlassen, weil sie in Wirts Chromosomen zu integrieren, um eine Anpassung Gene unkontrolliert auszudrücken. Selbst wenn virale Integration Transkriptionsfaktoren aktivieren oder inaktivieren Wirtsgene 8 kann eine unerwartete genetische Aberration und das Risiko von Tumorgenese 5,9 verursachen. Auf der anderen Seite wurden die direkte Einführung von Proteinen oder RNA in somatische Zellen berichtet, haben aber einige Nachteile, wie arbeitsintensiv, wiederholt transfbschnitt, und niedrige Niveau der Umprogrammierung 7,10. Auch episomal und nicht-integrierenden Adenovirus, Adeno-assoziiertes Virus und Plasmid-Vektoren sind noch relativ weniger effizient 11. Aus diesen Gründen ist es plausibel, Nicht-Integration Umprogrammierung Verfahren mit hoher Wirksamkeit iPSC Erzeugung und weniger genetische Anomalien zu wählen. In dieser Studie verwenden wir ein Sendai-Virus-basierten Neuprogrammierung. Diese Methode ist bekannt, dass in das Wirtsgenom nicht integrierten und konsequent produziert menschlichen iPS-Zellen ohne Transgen-Integrationen.

<table border="0" cellpadding="0" cellspacing="0" width="1109">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
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		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
			<td></td>
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efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

Vor ein paar Jahren, die Einrichtung des menschlichen induzierten pluripotenten Stammzellen (iPS-Zellen) in eine neue Ära in der Biomedizin eingeläutet. Mögliche Verwendung von menschlichen iPS-Zellen umfassen Modellierung Pathogenese der menschlichen genetischen Erkrankungen, autologe Zelltherapie nach der Gen-Korrektur und personalisierte Wirkstoff-Screening durch eine Quelle von Patienten-spezifischen und Symptom relevanten Zellen. Allerdings gibt es einige Hürden zu überwinden, wie die Beseitigung der verbleibenden Umprogrammierung Faktor Transgen-Expression nach menschlichen iPS-Zellen-Produktion. Noch wichtiger ist, könnte Rest Transgen-Expression in undifferenzierten menschlichen iPS richtige Differenzierungen behindern und verleiten die Auslegung von krankheitsrelevanten In-vitro-Phänotypen. Mit diesem Grund haben die Integration frei und / oder Transgen-freien menschlichen iPS-Zellen mit verschiedenen Methoden, wie z. B. Adenovirus, der piggyBac System, Minicircle-Vektor, episomaler Vektoren, direkte Lieferung Protein entwickelt und synthetisiert mRNA. Jedoch Effizienz reprogrammierung mit Integration freie Verfahren ist in den meisten Fällen sehr gering. Hier präsentieren wir eine Methode, um menschliche iPS-Zellen unter Verwendung von Sendai-Virus (RNA-Virus) auf der Basis Umprogrammierung System zu isolieren. Diese Umprogrammierung Methode zeigt, konsistente Ergebnisse und eine hohe Effizienz in kostengünstiger Weise.

Normalerweise infizierten Fibroblasten keine morphologischen Veränderungen in mehrere Tage nach Sendai-Virus-Transduktion zu zeigen, aber fünf Tage später, beginnen sie zu verschiedenen Formen (Abbildung 1) zu haben. Wie im rechten Teil der Abbildung 1 beschrieben, funktionieren Zellen nicht typisch Fibroblastenmorphologie mehr. Sie haben eine runde Form und größeren Kern als Zytoplasma. Auch bei Übertragungs wird in 80% zellulären Konfluenz durchgeführt wird, sieht es aus wie s...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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

Hier präsentieren wir unsere etablierte Methode zur menschlichen Körperzellen in gentechnikfreien menschlichen iPS-Zellen mit Sendai-Virus, das konsequente Ergebnis-und Effizienzsteigerung zeigt neu zu programmieren.

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Biology

22.2K Aufrufe.  Johns Hopkins University School of Medicine.  Hier präsentieren wir unsere etablierte Methode zur menschlichen Körperzellen in gentechnikfreien menschlichen iPS-Zellen mit Sendai-Virus, das konsequente Ergebnis-und Effizienzsteigerung zeigt neu zu programmieren. 

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

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 ...

Forschung

Biologie

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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, ...

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

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Schnelle und effiziente Erzeugung von Neuronen aus menschlichen pluripotenten Stammzellen in einem Multititre Platte Format

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

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 ...

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.