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>

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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><tbody><tr><td>CytoTune-iPS Reprogramming Kit</td><td>Invitrogen</td><td>A1378002</td><td></td></tr><tr><td>CF-6,MEFs, neomycin-resistant, mitomycin C treated&amp;nbsp;</td><td>Global stem</td><td>GSC-6105M</td><td>5 x 10&lt;sup&gt;5&lt;/sup&gt;/6 cm or 12.5 x 10&lt;sup&gt;5&lt;/sup&gt;/24-well plate</td></tr><tr><td>Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td><td>Invitrogen</td><td>25200114</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>Invitrogen</td><td>11330-032</td><td></td></tr><tr><td>24-well Cell Culture Plate, flat-bottom with lid</td><td>BD</td><td>353935</td><td></td></tr><tr><td>Y-27632</td><td>TOCRIS</td><td>1254</td><td>10 &amp;#956;M (Stock: 10 mM)</td></tr><tr><td>basic fibroblast growth factor</td><td>LIFE TECHNOLOGIES</td><td>PHG0263</td><td>10 ng (Stock : 100 ug)</td></tr><tr><td>Knock-out serum replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>Dulbecco&amp;#039;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td><td>Invitrogen</td><td>11965118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific Fermentas</td><td>SH30071.03</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>GIBCO</td><td>25030-081</td><td>1/100</td></tr><tr><td>MEM Non-Essential Amino Acids Solution, 100X</td><td>LIFE TECHNOLOGIES</td><td>11140050</td><td>1/100</td></tr><tr><td>2-Mercaptoethanol (1,000X), liquid</td><td>GIBCO</td><td>21985023</td><td>1/1,000</td></tr><tr><td>Hausser Phase Contrast Hemacytometers</td><td>Hausser Scientific</td><td>02-671-54</td><td></td></tr><tr><td>EmbryoMax 0.1% Gelatin Solution</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>SSEA-4</td><td>DSHB</td><td>MC-813-70</td><td>1/200</td></tr><tr><td>anti-Tra-1-81</td><td>Cell Signaling</td><td>4745S</td><td>1/200</td></tr><tr><td>mouse monoclonal Oct4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>1/1,000</td></tr><tr><td>Nanog</td><td>R&amp;amp;D</td><td>AF1997</td><td>1/1,000</td></tr><tr><td>Alexa Flouor 488 goat anti-mouse</td><td>Invitrogen</td><td>948492</td><td>1/2,000</td></tr><tr><td>DPBS (Dulbecco&amp;#039;s Phosphate-Buffered Saline), 1X without calcium &amp;amp; magnesium</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit</td><td>QIAGEN</td><td>205313</td><td></td></tr><tr><td>PCR Master Mix [2X]</td><td>Thermo Scientific Fermentas</td><td>K0171</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>picking hood</td><td>NuAire</td><td>NU-301</td><td></td></tr><tr><td>dissecting scope&amp;nbsp;</td><td>Nikon</td><td>SMZ745</td><td></td></tr></tbody></table>

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

22.5K 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 from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Erzeugung von induzierten pluripotenten Stammzellen aus Gefrorene Buffy Coats mit Non-Integration episomalen Plasmiden

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Forschung

Entwicklungsbiologie

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen peripheren T-Zellen Mit Sendai Virus in Feeder-freien Bedingungen

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Die Erzeugung von induzierten pluripotenten Stammzellen aus menschlichen Melanom-Tumor-infiltrierenden Lymphozyten

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induzierte pluripotente Stammzellen Erzeugung von Blutzellen mit Sendai-Virus und Zentrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Die Erzeugung von Integration freien induzierte pluripotente Stammzellen aus menschlichen peripheren einkernigen Blutzellen Mit Episomale Vektoren

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

Biologie

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Generierung induzierter neuronaler Stammzellen aus peripheren mononukleären Zellen und Differenzierung zu dopaminergen Neuronenvorstufen für Transplantationsstudien

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Effiziente Erzeugung und Bearbeitung von Feeder-freie IPSCs aus menschlichen Zellen der Bauchspeicheldrüse mit dem CRISPR-Cas9-System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Biotechnik

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 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, ...