Diese Arbeit wurde von Friedreich-Ataxie Research Alliance und einer Pilot-Zuschuss von Arnold Family Foundation und dem Zentrum für Stammzellen und Entwicklungsbiologie am MD Anderson Cancer Center unterstützt.

1. Virus Produktion und Transduktion <ol><li> Tafel Phoenix Ampho-Zellen bei einer Dichte von ~ 7-8x10 6 pro 10 cm Platte in 10 ml DMEM-Medium (DMEM hohe Glucose, 10% Hitze-inaktiviertem FBS, 2 mM L-Glutamin, keine Antibiotika). Platz im Inkubator und Kultur über Nacht bei 37 ° C, 5% CO 2. </li><li> Am nächsten Tag Transfektion Phoenix Zellen unter Verwendung von 12 ug einem Vektor, der entweder Oct3 / 4, Sox2, Klf4, c-myc oder GFP-Gen (Addgene Plasmide 17.217, 17.218, 17.219, 17.220) und 35 ul Fugene 6. Planen Transfektionsgemisch in 500 ul DMEM-Medium (DMEM hohe Glucose, 2 mM L-Glutamin, nicht FBS und Antibiotika). Inkubieren 20 min. Vorsichtig pipettieren DNA-Komplexe in die 10 cm-Platten mit 70-80% konfluenten Phoenix-Zellen. </li><li> Ersetzen Medien 6 - 8 h nach der Transfektion mit DMEM-Medium (DMEM high glucose, 10% FBS hitzeinaktiviertem, 2 mM L-Glutamin) mit Penicillin und Streptomycin. </li><li> Anschließend sammeln Virus-haltigen Medien 4-mal in 12 hIntervalle, kombinieren alle Teile und durch ein 0,45 um-Filter, um abgelösten Zellen und Ablagerungen (Medien mit viralen Partikeln kann im Kühlschrank bei 2 Wochen, ohne dass infektiöse Aktivität gehalten werden, jedoch Einfrieren wird nicht empfohlen) entfernen. </li><li> Für retrovirale Transduktion, Platte humanen adulten Fibroblasten (Passage 10, Coriell Laboratories) aus einem gefrorenen Lager 24 h vor dem letzten Tag des retroviralen Mediensammlung. Seed die Zellen auf 6-Loch-Platten abgedeckt Gelatine mit einer Dichte 1x10 5 Zellen pro Vertiefung in DMEM hohe Glucose, 10% FBS, 2 mM L-Glutamin, Penicillin, Streptomycin und nicht-essentiellen Aminosäuren. </li><li> Am nächsten Tag Ersatz DMEM-Medium mit Medien, die viralen Partikel aus Phoenix-Zellen erhalten. Fügen Sie viralen Medien (1 ml jeder Oct4, Sox2, Klf4, und c-myc-Medien, insgesamt 4 ml) mit 6 pg / ml Polybren in jedes Well der 6-Well-Platte der Fibroblasten-Kulturen und Zentrifuge ergänzt bei 1600g für 1h bei 20 ° C Etwa 12 h after die Transduktion, ersetzen Sie die viralen Medien mit Fibroblasten-Kulturmedium. Führen Virusinfektion und Zentrifugation 3-mal in 24 h Intervallen. </li><li> Kultur Fibroblasten in DMEM-Medium für 48 h nach der letzten Infektion. Die Effizienz der Infektion durch parallele Transduktionen mit einem retroviralen Medien, die GFP exprimieren. 1 zeigt die Effizienz der Zentrifugation erleichtert-retroviralen Infektion von humanen Fibroblasten. </li></ol> 2. Reprogrammierung <ol><li> Planen 6-Well-Platten mit γ-bestrahlten MEF-Feeder-Schichten durch Impfen MEF-Zellen bei einer Dichte von ~ 2x10 5 Zellen pro Vertiefung der Gelatine behandelten 6-Well-Platte in Fibroblasten-Wachstumsfaktor Medium. Am folgenden Tag aufgeteilt infizierten menschlichen Fibroblasten unter Verwendung von 0,05% Trypsin / EDTA und entkernen mit einer Dichte von ~ 1.2x10 4 pro auch in der Fibroblasten Wachstumsmedium. </li><li> Am nächsten Tag ersetzen Medien mit hES-Medien (DMEM/F12, 20% Knockout Serumersatz, nicht-essentielle Aminosäuren, penicillin / Streptomycin, 2 mM L-Glutamin, 0,1 mM β-Mercaptoethanol, 20 ng / ml basalen Fibroblast Growth Factor (bFGF), ergänzt mit 0,5 mM Natriumbutyrat für die ersten 7 Tage nach Umprogrammierung. Ändern Medien täglich. </li><li> Überwachen morphologischen Veränderungen in transduzierten Zellen täglich. Kolonien von HIPS-ähnlichen Zellen beginnt zu entstehen ca. 5 - 10 Tage nach der Übertragung der transduzierten Fibroblasten auf Feeder-Zellen. Die hiPSC Kolonien sind bereit, rund 14 isoliert werden - 28 Tage nach dem Ausplattieren sie auf den MEF-Feeder-Zellen. </li></ol> 3. Isolierung von Kolonien hiPSC <ol><li> Einen Tag vor Aufnahme der hiPSC Kolonien herzustellen 24-Well-Platten mit γ-bestrahlten MEF Feeder-Zellen (~ 4x10 4 Zellen pro Vertiefung) in Fibroblasten Wachstumsmedium. In diesem Stadium hiPSC Kolonien können auch auf Feeder-freien Kultur mit Matrigel und mTeSR1 Medium übertragen werden. </li><li> Vor der Isolierung des hiPSC Kolonien herzustellen MEF Platten durch Entfernen Fibroblasten Wachstum Medium und Spülen mit PBS MEFs, um Spuren von FBS zu entfernen. Anschließend werden 0,5 ml HES Medium (oder mTeSR1 Mediums im Falle von Matrigel beschichteten Vertiefungen) mit 10 pM ROCK-Inhibitor Y27632 in jede Vertiefung 7,8. </li><li> Falls erforderlich, die rund um die Fibroblasten hiPSC Kolonien mit einem 21-Gauge-Nadel unter einem Mikroskop in einem Abzug mit laminarer Strömung (2A - C) angebracht ist. Spülen Sie die Platten mit PBS und fügen frische hES-Datenträger mit dem Tra-1-81 StainAlive spezifischen Antikörper (1:200, Stemgent). Nach 30 min., Ersetzen Sie Medien, die den Antikörper mit frischen hES-Medien mit 10 uM ROCK-Inhibitor ergänzt. Untersuchen Sie die Platten unter dem Fluoreszenzmikroskop und Tra-Marke von 1 bis 81 positive Kolonien mit Hilfe einer objektiven Marker (2D). </li><li> Schneiden Sie die Tra-1-81 positive hiPSC Kolonien unter dem Mikroskop in einer Laminar-Haube, in mehrere kleine Stücke mit einer 21-Gauge-Nadel wie in 2E gezeigt und F. </li><li> Verwendung einer automatischen Pipette (P200) Transfer Fragmente von hiPSC Kolonien in individual Vertiefungen der 24-Well-Platte mit MEFs oder Matrigel. Vermeiden Sie die Übertragung nicht-HIPS-Zellen. Legen Sie die Platten in einem 37 ° C, 5% CO 2-Inkubator und lassen Sie die hiPSC Kolonien für 24-36 h zu befestigen. </li><li> Ändern HES oder mTeSR1 (für Kolonien auf Matrigel gewachsen) Medien täglich. Die Kolonien mit hiPSC richtige HES-Morphologie sichtbar 48 h nach der ersten Übertragung auf einer 24-Well-Platte. </li><li> Manuelles Durchgang hiPSC Kolonien alle 6 - 8 Tage auf einer 12-Well und anschließend auf einem 6-Well-Platte. </li><li> Nach klonale Expansion und zur Festlegung hiPSC Linien, zu analysieren Expression von Markern für Pluripotenz TRA-1-60, SSEA-4, Oct3 / 4, Sox2 und Nanog Verwendung von Immunocytochemie wie in 3 gezeigt. Bewerten genomischen Integrität und Differenzierungspotential der erhaltenen Linien mit Karyotyp und Teratombildung Analysen 6,9. </li></ol> 4. Repräsentative Ergebnisse Effiziente Transduktion mit Retrovirus-haltigen Medien is die für den erfolgreichen Reprogrammierung. Es wird empfohlen, den gesamten Transfektion / Infektion Verfahren durchzuführen unter Verwendung eines GFP-exprimierenden Virus jede einzelne Anpassung Experiment, um die Effizienz zu überwachen, wie in 1 gezeigt. Der Titer des GFP-exprimierenden Virus bestimmt, wie in 10 beschrieben, durch die Transduktion von humanen Fibroblasten mit nicht-konzentrierten viralen Medien lag typischerweise im Bereich von 0,5 - 5 x 10 7 Viruspartikeln pro ml (vp / ml). Fibroblasten ändern Morphologie bereits 2 Tage nach der letzten Infektion. Trypsiniert humanen Fibroblasten sollte sorgfältig gezählt werden, bevor sie auf die Aussaat MEF Feeder-Zellen. Es ist zu Samenzellen bei 3 verschiedenen Dichten (6x10 3, 4 1.2x10, 2.5x10 4 pro einzelnes Loch einer 6-Well-Platte) empfohlen, da jeder Zelllinie zeigt verschiedene charakteristische Wachstum und Aussaatdichte ist entscheidend für die Effizienz der Reprogrammierung. Natriumbutyrat für die Verwendungersten 7 - 14 Tagen der Umprogrammierung erhöht die Effizienz der Bildung hiPSC ca. 5 fache. Häufig, insbesondere wenn infizierte Fibroblasten in höherer Dichte ausgesät wurden, können die Fibroblasten-ähnliche Zellen überwachsen eine Kulturschale und decken hiPSC Kolonien in 2A gezeigt. In diesem Fall kann die Fibroblasten-Schicht vorsichtig angehoben werden und entfernt, um hiPSC Kolonien (2B) aufzudecken. Anschließend sollte hiPSC Kolonien mit hES-Medien gespült werden und gefärbt mit Tra-1-81 Antikörper. Abhängig von den Fibroblasten-Zellen, ca. 20 - 40% der Kolonien demonstriert mit IPSC-Morphologie nicht mit Tra-1-81 Antikörper färben. 24h - identifiziert hiPSC Kolonien können aus einer Platte innerhalb der nächsten 12 übertragen werden. Längerer Inkubation wird in schnelleren Differenzierung von hiPSCs führen. Die Kolonien können entweder manuell auf Feeder-Zellen oder MEF Matrigel-beschichteten Platten passagiert werden. Nach der Expansion etabliert hiPSC Klone sollte getestet mit Immunzytochemie (ICC) für die Expression von pluripoten werdency-Marker wie in Abbildung 3 gezeigt. Zusätzlich sollte eine detaillierte molekulare Charakterisierung der erzeugten iPS-Zelllinien umfassen: Analysen der Pluripotenz Genexpression mittels RT-PCR, Demonstration der DNA-Demethylierung bei den Promotoren von Pluripotenz-Gene und-analysen der Transgene Silencing-9. <img alt="1" src="/files/ftp_upload/3416/3416fig1.jpg" /> Abbildung 1. Die Effizienz der virale Transduktion unter Verwendung GFP-exprimierenden Retrovirus ist. (A, B) Humane adulte Fibroblasten, die von Friedreich-Ataxie Patienten abgeleitet (GM03665, Coriell Repository) wurden sichtbar nach zwei aufeinander folgenden Infektionen mit GFP retroviralen Medien. (C, D) Humane Fibroblasten wurden mit der gleichen Charge der GFP retroviralen Medien durch Zentrifugation der Zellen direkt auf den Platten mit 6 Vertiefungen für 1 Stunde bei 1600g gefolgt infiziert. Die Bilder wurden 48h nach der Infektion eingefangen. <img alt= &quot;Bild 2&quot; src = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; Abbildung 2. Identifizierung und Isolierung von hiPSC Kolonien. (A) Phasenkontrast-Bild einer Platte mit hiPSCs von Fibroblasten umgeben. Die Zellen wurden für 21 Tage auf hES Medien kultiviert. (B, C) Das gleiche hiPSC Kolonie nach dem Entfernen des umgebenden Fibroblasten-Schicht. (D) korrekt neu programmiert hiPSC Kolonien, die von Live-Färbung mit Tra-Oberfläche 1-81 Marker-Antikörper identifiziert werden, schneiden mit einer sterilen Kanüle (E, F) und in separaten Kammern einer 24-Well-Platte. <img alt="Abbildung 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> 3. Expression der Pluripotenz Marker Oct3 / 4, Nanog, Sox2, SSEA4 und Tra-1-60 in hiPSCs wurde durch Immunzytochemie bestimmt.

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Die Autoren haben nichts zu offenbaren.

Studieren menschlicher Krankheiten, insbesondere neurologische und neurodegenerative, wurde eine besondere Herausforderung aufgrund der Unzugänglichkeit der angemessene personelle zellulären Modellen. Die Fähigkeit, leicht erhältlich Körperzellen in induzierte pluripotente Stammzellen umprogrammieren und das Potenzial, um sie in verschiedenen Zelltypen differenzieren eröffnet die Möglichkeit, zelluläre Modelle von genetischen Krankheiten zu schaffen. Darüber hinaus halten IPSCs ein großes Versprechen für die ...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10828 </td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Natriumbutyrat </td><td> Sigma </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81-Antikörper </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 Antikörper </td><td> Santa Cruz </td><td> SC-8628 </td></tr><tr><td> Nanog Antikörper </td><td> Cell Signaling Technology </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60-Antikörper </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 Antikörper </td><td> Cell Signaling Technology </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFs </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> Ziel-Marker </td><td> Nikon </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td> StemCell Technologies </td><td> 05850 </td></tr><tr><td> β-Mercaptoethanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> Polybren </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Objekt-Marker </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Hier präsentieren wir ein Protokoll der Reprogrammierung erwachsener Mensch menschlichen Fibroblasten in induzierte pluripotente Stammzellen (hiPSC) mit retroviralen Vektoren kodieren Oct3 / 4, Sox2, Klf4 und c-Myc (OSKM) in Gegenwart von Natriumbutyrat 1-3. Wir verwendeten diese Methode, um spät Passage neu zu programmieren (&gt; P10) humanen adulten Fibroblasten von Friedreich-Ataxie Patienten abgeleitet (GM03665, Coriell Repository). Die Umprogrammierung beinhaltet hoch effiziente Transduktion Protokoll unter Verwendung von Fibroblasten wiederholte Zentrifugation in Gegenwart von Virus-enthaltenden Medien. Die modifizierte hiPSC Kolonien identifiziert wurden unter Verwendung lebender Immunfärbung für Tra-1-81, ein Oberflächenmarker von pluripotenten Zellen, aus nicht-programmiert Fibroblasten getrennt und manuell passagiert 4,5. Diese hiPSC wurden dann auf Matrigel Platten übertragen und aufgewachsen in Feeder-freien Bedingungen, direkt aus der Umprogrammierung Platte. Ab dem ersten Durchgang, demonstrieren hiPSC charakteristische Kolonien hES-like Morphologie. Unter Verwendung dieses Protokolls mehr als 70% der ausgewählten Kolonien erfolgreich ausgeweitet und festgesetzt werden, in Zelllinien. Die etablierten hiPSC Linien angezeigt charakteristischen Markern für Pluripotenz einschließlich Oberflächenmarker TRA-1-60 und SSEA-4, sowie nukleare Marker Oct3 / 4, Sox2 und Nanog. Das hier vorgestellte Protokoll wurde eingerichtet und getestet mit adulten Fibroblasten von Friedreich-Ataxie Patienten und Kontrollpersonen 6, menschlichen Neugeborenen Fibroblasten, sowie menschliche Keratinozyten erhalten.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Wir präsentieren ein Protokoll für die effiziente Reprogrammierung von menschlichen Körperzellen in die menschlichen induzierten pluripotenten Stammzellen (hiPSC) mit retroviralen Vektoren kodieren Oct3 / 4, Sox2, Klf4 und c-Myc (OSKM) und Identifizierung von richtig umprogrammiert hiPSC durch Live-Färbung mit Tra- 1-81 Antikörper ist.

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 ...

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

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19.8K Aufrufe.  University of Texas M.D. Anderson Cancer Center. Wir präsentieren ein Protokoll für die effiziente Reprogrammierung von menschlichen Körperzellen in die menschlichen induzierten pluripotenten Stammzellen (hiPSC) mit retroviralen Vektoren kodieren Oct3 / 4, Sox2, Klf4 und c-Myc (OSKM) und Identifizierung von richtig umprogrammiert hiPSC durch Live-Färbung mit Tra- 1-81 Antikörper ist.

Serie: Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

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, ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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 ...

Verwendung von Hautbiopsien zur Erzeugung von patienteneigenen Podozyten

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Autor im Rampenlicht: Generierung von patienteneigenen Podozyten aus Hautbiopsien  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Generierung von patienteneigenen Podozyten aus Hautbiopsien

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...