Ce travail a été soutenu par l&#39;ataxie de Friedreich Alliance de recherche et une subvention pilote de la famille Arnold Fondation et le Centre pour les cellules souches et la biologie du développement au MD Anderson Cancer Center.

1. La production de virus et de la transduction <ol><li> Plate Phoenix cellules Ampho à une densité de ~ 7-8x10 6 pour plaque de 10 cm dans 10 ml de milieu DMEM (DMEM taux élevé de glucose, 10% de la chaleur FBS inactivé, 2 mM de L-glutamine, pas d&#39;antibiotiques). Placer dans l&#39;incubateur et de la culture une nuit à 37 ° C, 5% de CO 2. </li><li> Le jour suivant, la transfection de cellules en utilisant Phoenix 12 ug d&#39;un vecteur codant soit Oct3 / 4, Sox2, Klf4, c-myc, ou GFP gène (Addgene plasmides 17217, 17218, 17219, 17220) et 35 ul Fugène 6. Préparer le mélange de transfection dans 500 pi de milieu DMEM (DMEM taux élevé de glucose, 2 mM de L-glutamine, sans FBS et des antibiotiques). Incuber 20 min. Doucement pipeter complexes d&#39;ADN dans les plaques de 10 cm contenant 70-80% confluentes cellules Phoenix. </li><li> Remplacez le support de 6 à 8 h post-transfection avec du milieu DMEM (DMEM taux élevé de glucose, 10% de la chaleur FBS inactivé, 2 mM de L-glutamine) contenant de la pénicilline et la streptomycine. </li><li> Par la suite, de recueillir contenant le virus des médias 4 fois en 12 heuresintervalles, de combiner toutes les parties et filtré en utilisant un filtre de 0,45 um pour éliminer les cellules individuelles et les débris (médias contenant des particules virales peuvent être conservés dans le réfrigérateur pendant 2 semaines sans perdre activité infectieuse, mais la congélation n&#39;est pas recommandée). </li><li> Pour rétroviraux fibroblastes de transduction de la plaque, les adultes de l&#39;homme (passage 10, Coriell Laboratories) à partir d&#39;un stock de 24 h congelés avant le dernier jour de la collecte des médias rétroviral. Ensemencer les cellules sur 6 et la gélatine des plaques recouvertes de les 1x10 densité 5 cellules par puits dans du DMEM taux élevé de glucose, 10% de FBS, 2 mM de L-glutamine, pénicilline, la streptomycine et non-acides aminés essentiels. </li><li> Le substitut lendemain milieu DMEM avec les médias contenant des particules virales obtenues à partir de cellules de Phoenix. Ajouter des médias viraux (1 ml de chaque Oct4, Sox2, Klf4, et c-myc médias, 4 ml au total) additionnée de 6 mg / ml de polybrène dans chaque puits de la plaque à 6 puits des cultures de fibroblastes et de centrifugeuses à 1600g pour 1h à 20 ° C. Environ 12 h after la transduction, de remplacer les médias virales avec un milieu de culture des fibroblastes. Effectuer une infection virale et la centrifugation 3 fois à intervalles de 24 h. </li><li> Fibroblastes Culture en milieu DMEM pour 48 h après l&#39;infection. Dernière Vérifier l&#39;efficacité de l&#39;infection par transductions parallèles avec un média rétroviraux exprimant la GFP. La figure 1 illustre l&#39;efficacité de la centrifugation facilitée par infection rétrovirale des fibroblastes humains. </li></ol> 2. Reprogrammation <ol><li> Préparer plaques à 6 puits avec γ-irradiés couches nourricières du MEF en ensemençant les cellules MEF à une densité de ~ 2x10 5 cellules par puits de la gélatine traitée plaque de 6 puits dans le milieu de croissance des fibroblastes. Le lendemain, diviser infectés fibroblastes humains en utilisant 0,05% de trypsine / EDTA et les épépiner à la densité de ~ 1.2x10 4 par puits dans le milieu de croissance des fibroblastes. </li><li> Le lendemain, remplacer le support avec les médias hES (DMEM/F12, remplacement 20% de sérum Knockout, non-acides aminés essentiels, pénicillesn / streptomycine, 2 mM de L-glutamine, 0,1 mM β-mercaptoéthanol, 20 ng / ml de facteur de croissance des fibroblastes de base (bFGF), supplémenté avec 0,5 mM de butyrate de sodium pour les 7 premiers jours de la reprogrammation. Changer les médias tous les jours. </li><li> Surveiller les changements morphologiques dans les cellules transduites par jour. Les colonies de cellules de type HIPS va commencer à émerger d&#39;environ 5 - 10 jours après le transfert des fibroblastes transduits sur des cellules nourricières. Les colonies hiPSC sont prêts à être isolé d&#39;environ 14 - 28 jours après leur étalement sur les cellules nourricières MEF. </li></ol> 3. Isolement des colonies hiPSC <ol><li> Une journée avant de reprendre les colonies hiPSC préparer des plaques 24 puits avec γ-irradiés cellules nourricières du MEF (environ 4x10 4 cellules par puits) dans la croissance des fibroblastes à moyen terme. A ce stade colonies hiPSC peuvent également être transférées à la culture chargeur gratuitement en utilisant le Matrigel et moyennes mTeSR1. </li><li> Avant l&#39;isolement des colonies hiPSC préparer les plaques du MEF en supprimant la croissance des fibroblastes Medium et le rinçage avec du PBS MEF pour éliminer les traces de FBS. Par la suite, ajoutez 0,5 ml de milieu de hES (ou mTeSR1 moyenne dans le cas de Matrigel puits revêtus) avec 10 inhibiteur de ROCK uM Y27632 à chaque 7,8 bien. </li><li> Si nécessaire, retirez les fibroblastes entourant les colonies hiPSC avec une aiguille de calibre 21 sous un microscope monté dans une hotte à flux laminaire (Figure 2A - C). Rincer les plaques avec du PBS et ajouter de nouvelles des médias hES contenant l&#39;anticorps Tra-1-81 StainAlive spécifique (1:200, Stemgent). Après 30 min., De remplacer le support contenant l&#39;anticorps avec les médias hES frais supplémenté avec 10 uM inhibiteur de ROCK. Examiner les plaques sous la marque de microscope à fluorescence et TRA-1-81 colonies positives en utilisant un marqueur objectif (figure 2D). </li><li> Couper les Tra-1-81 colonies positives hiPSC sous un microscope dans une hotte laminaire, en plusieurs petits morceaux en utilisant une aiguille de calibre 21 comme indiqué dans la figure 2E et F. </li><li> En utilisant une pipette de transfert automatique des fragments (P200) de colonies hiPSC en indpuits ividual de la plaque de 24 puits avec MEF ou Matrigel. Éviter de transférer des non-hanches cellules. Placez les plaques à 37 ° C, 5% de CO 2 incubateur et permettre aux colonies hiPSC se fixer pendant 24-36 h. </li><li> Changement HES ou mTeSR1 (pour les colonies cultivées sur Matrigel) les médias tous les jours. Les colonies hiPSC avec correcte HES-morphologie sera visible 48 h après le transfert initial sur une plaque de 24 puits. </li><li> Manuellement le passage des colonies hiPSC tous les 6 à 8 jours SUR UNE de 12 puits et ensuite sur une plaque à 6 puits. </li><li> Après expansion clonale et d&#39;établir les lignes hiPSC, analyser l&#39;expression des marqueurs de pluripotence TRA-1-60, AESS-4, Oct3 / 4, Sox2 et Nanog utilisant immunocytochimie comme le montre la figure 3. Évaluer l&#39;intégrité du génome et le potentiel de différenciation des lignées obtenues à l&#39;aide du caryotype et analyses formation de tératome 6,9. </li></ol> 4. Les résultats représentatifs Transduction efficace avec un rétrovirus contenant les médias is critique pour reprogrammation réussie. Il est recommandé de procéder à l&#39;ensemble de la transfection / infection en utilisant une procédure de GFP exprimer virus chaque expérience unique reprogrammation de surveiller l&#39;efficacité comme le montre la figure 1. Le titre du virus exprimant la GFP déterminée, tel que décrit dans 10, dans la transduction des fibroblastes humains en utilisant non concentrées support virales était généralement dans la plage de 0,5 à 5 x 10 7 particules virales par ml (pv / ml). Fibroblastes changer la morphologie dès 2 jours après la dernière infection. Trypsinisées fibroblastes humains doivent être soigneusement comptés avant de les semer sur les cellules nourricières du MEF. Il est recommandé de cellules de semence à 3 densités différentes (6x10 3, 1.2x10 4, 2,5.10 4 pour seul puits d&#39;une plaque à 6 puits) puisque chaque lignée cellulaire démontre caractéristique de croissance et de densité différentes semis est essentielle pour l&#39;efficacité de la reprogrammation. Le butyrate de sodium utilisé pour leinitial de 7 - 14 jours de la reprogrammation augmente l&#39;efficacité de la formation hiPSC environ 5 fois. Souvent, surtout quand les fibroblastes infectés ont été ensemencées à une densité plus élevée, les cellules fibroblast-like peut recouvrir une boîte de culture et de couvrir les colonies hiPSC comme le montre la figure 2A. Dans ce cas, la couche de fibroblastes peut être levée en douceur et enlevé pour découvrir les colonies hiPSC (figure 2B). Par la suite, les colonies hiPSC doit être rincée avec hES médias et colorées avec Tra-1-81 anticorps. Selon les cellules de fibroblastes, d&#39;environ 20 à 40% des colonies démontrant avec iPSC-morphologie ne se colorent pas avec Tra-1-81 anticorps. Identifiés colonies hiPSC peut être transférée d&#39;une plaque dans les 12 prochaines - 24h. Une incubation prolongée se traduira par une différenciation rapide des hiPSCs. Les colonies peuvent être repiquées sur manuellement, soit des cellules nourricières du MEF ou Matrigel plaques enduites. Après l&#39;expansion établie clones hiPSC devrait être testé à l&#39;aide immunocytochimie (ICC) pour l&#39;expression de pluripotency marqueurs comme l&#39;a démontré dans la figure 3. En outre, une caractérisation moléculaire détaillée des lignes générées iPS cellules devraient inclure: l&#39;analyse de l&#39;expression du gène pluripotence par RT-PCR, la démonstration de la déméthylation de l&#39;ADN dans les promoteurs des gènes de pluripotence et des analyses des transgènes silencing 9. <img alt="Figure 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> Figure 1. L&#39;efficacité de la transduction virale déterminée à l&#39;aide rétrovirus GFP exprimer. (A, B) des fibroblastes adultes humains provenant des patients l&#39;ataxie de Friedreich (GM03665, Coriell Repository) ont été visualisées après deux infections consécutives à des médias GFP rétroviraux. (C, D) Des fibroblastes humains ont été infectés avec le même lot des médias GFP rétroviraux suivis par centrifugation des cellules directement sur les plaques de 6 puits pour 1h à 1600g. Les images ont été capturées 48h après l&#39;infection. <img alt= &quot;Figure 2&quot; src = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot; /&gt; Figure 2. Identification et isolement de colonies hiPSC. (A) l&#39;image en contraste de phase d&#39;une plaque contenant hiPSCs entourés par les fibroblastes. Les cellules ont été cultivées pendant 21 jours sur hES médias. (B, C) de la colonie même hiPSC après enlèvement de la couche de fibroblastes entourant. (D) correctement reprogrammées colonies hiPSC sont identifiés par la coloration en direct avec un anticorps marqueur Tra-1-81 de surface, découpé à l&#39;aide d&#39;une aiguille stérile (E, F) et transféré à des puits séparés d&#39;une plaque de 24 puits. <img alt="Figure 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> Figure 3. Expression des marqueurs de pluripotence spécifiques Oct3 / 4, Nanog, Sox2, SSEA4 et Tra-1-60 en hiPSCs a été déterminée par immunocytochimie.

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Les auteurs n&#39;ont rien à révéler.

Étude des maladies humaines, notamment neurologiques et neurodégénératives, a été particulièrement difficile en raison de l&#39;inaccessibilité de l&#39;homme des modèles cellulaires adéquats. La capacité de reprogrammer des cellules somatiques faciles à obtenir en cellules souches pluripotentes induites et la possibilité de les différencier en divers types cellulaires a ouvert une possibilité de créer des modèles cellulaires de maladies génétiques. En outre, CSPi tenir une grande promesse pour l&#39;...

<table border="1"><tbody><tr><td> Réactif </td><td> Entreprise </td><td> Le numéro de catalogue </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> Non-essentiels acides aminés </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Le butyrate de sodium </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 anticorps </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 d&#39;anticorps </td><td> Santa Cruz </td><td> sc-8628 </td></tr><tr><td> Nanog anticorps </td><td> Cell Technology de signalisation </td><td> 4903S &lt;/ Td&gt; </tr><tr><td> Tra-1-60 anticorps </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 anticorps </td><td> Cell Technology de signalisation </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEF </td><td> Globalstem </td><td> CGC-6201G </td></tr><tr><td> Objectif marqueur </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-mercaptoéthanol </td><td> Sigma </td><td> M7522 </td></tr><tr><td> Fugène 6 </td><td> Roche </td><td> 11814443001 </td></tr><tr><td> polybrène </td><td> Sigma </td><td> H9268 </td></tr><tr><td> Marqueur d&#39;objet </td><td> Nikon </td><td> MBW10010 </td></tr></tbody></table>

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

On présente ici un protocole de reprogrammation fibroblastes humains adultes humaines en cellules souches pluripotentes induites (hiPSC) en utilisant des vecteurs rétroviraux encodant Oct3 / 4, Sox2, Klf4 et c-myc (OSKM) en présence de sodium 1-3 butyrate. Nous avons utilisé cette méthode pour reprogrammer passage tardif (&gt; p10) fibroblastes adultes humaines dérivées de patients l&#39;ataxie de Friedreich (GM03665, Coriell Repository). L&#39;approche reprogrammation comprend protocole de transduction hautement efficace en utilisant une centrifugation répétitif de fibroblastes en présence d&#39;un milieu contenant de virus. Les colonies hiPSC reprogrammées ont été identifiés à l&#39;aide immunomarquage direct pour Tra-1-81, un marqueur de surface de cellules pluripotentes, séparés à partir de fibroblastes non-reprogrammés manuellement et passages 4,5. Ces hiPSC ont ensuite été transférés sur des plaques de Matrigel et cultivés dans des conditions exemptes de desserte, directement à partir de la plaque de reprogrammation. A partir du premier passage, les colonies hiPSC démontrer caractéristique HES-lmorphologie ike. En utilisant ce protocole, plus de 70% des colonies sélectionnées peuvent être élargi avec succès et mis en place dans des lignées cellulaires. Les lignes établies hiPSC affiché caractéristiques des marqueurs de pluripotence, y compris les marqueurs de surface TRA-1-60 et SSEA-4, ainsi que des marqueurs nucléaires Oct3 / 4, Sox2 et Nanog. Le protocole présenté ici a été établi et testé à l&#39;aide des fibroblastes adultes obtenus chez des patients ataxie de Friedreich et de contrôle des individus, des fibroblastes humains 6 nouveau-nés, ainsi que les kératinocytes humains.

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

Nous présentons un protocole de reprogrammation efficace des cellules somatiques humaines dans l&#39;homme cellules souches pluripotentes induites (hiPSC) en utilisant des vecteurs rétroviraux codant Oct3 / 4, Sox2, Klf4 et c-myc (OSKM) et l&#39;identification des hiPSC correctement reprogrammé par coloration en direct avec des Tra 1-81 anticorps.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

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 vues.  University of Texas M.D. Anderson Cancer Center. Nous présentons un protocole de reprogrammation efficace des cellules somatiques humaines dans l'homme cellules souches pluripotentes induites (hiPSC) en utilisant des vecteurs rétroviraux codant Oct3 / 4, Sox2, Klf4 et c-myc (OSKM) et l'identification des hiPSC correctement reprogrammé par coloration en direct avec des Tra 1-81 anticorps.

Vidéo: 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.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes humains avec les lentivirus TF Stemgent Homme Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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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.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

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.

Transfection et Nucleofecting homme cellules souches pluripotentes induites

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

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.

Reprogrammation des cellules somatiques humaines dans cellules souches pluripotentes induites (CISP) Utilisation vecteur rétroviral avec la 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.

Génération de souris dérivées de cellules souches pluripotentes induites

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.

Génération de l&#39;homme cellules souches pluripotentes induites à partir de sang périphérique en utilisant le vecteur lentiviral STEMCCA

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Génération de cardiomyocytes humains: Un protocole de différenciation des induits des cellules souches pluripotentes humaines chargeur sans

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.

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec 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 ...

Utilisation de biopsies cutanées pour générer des podocytes dérivés de patients

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.

Pleins feux sur l’auteur : Génération de podocytes dérivés de patients à partir de biopsies cutanées  

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.

Génération de podocytes dérivés de patients à partir de biopsies cutanées

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