1. Reprogrammation <ol><li> Cellules BJ semences à une densité de 1 x 10 5 cellules par puits d&#39;une plaque à 6 puits. Culture des cellules dans 2 ml de milieu de croissance (450 ml EMEM supplémenté avec 50 ml de FBS ES qualifié, 5 ml 10 mM non acides aminés essentiels, 5 ml de pénicilline / streptomycine, 5 ml de 200 mM de L-glutamine et 0,9 ml β 55 mm -mercaptoéthanol) nuit à 37 ° C et 5% de CO 2. </li><li> Le même jour, commencer à préparer les plaques d&#39;alimentation du MEF en ajoutant 0,1% de gélatine diluée dans de l&#39;eau sur une plaque de 6 puits et incuber pendant une nuit à 37 ° C et CO 2 5%. </li><li> Après incubation, éliminer le milieu à partir des cellules BJ et ajouter 2 ml milieu de croissance complété avec 6 pg / ml de polybrène, 500 ul hOct4-lentivirus, 50 ul hSox2-lentivirus, 50 ul hNanog-lentivirus et 50 pi hLin28-lentivirus dans chaque puits . Secouez doucement la plaque pour assurer un revêtement uniforme du milieu. Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Le même jour, enlever 1 flacon de cellules MEF FC-1 de l&#39;azote liquide et de dégel. Retirer le liquide des puits de gélatine enduit (voir étape 1.2) et ajouter des cellules nourricières du MEF à une densité de 0,2 x 10 5 cellules / puits. Le volume total par puits devrait être 2ml de cellules dans un milieu de croissance du MEF (450 ml de DMEM supplémenté avec 50 FBS ml et 5 ml non acides aminés essentiels). </li><li> Le lendemain, détachez les cellules BJ avec 0,05% de trypsine / EDTA et centrifuger à 200 xg pendant 5 minutes. Aspirer le moyen et remettre en suspension dans un milieu de croissance. Retirer de la plaque de support chargeur de MEF (voir étape 1.4) et ajouter 2 ml / puits de la suspension cellulaire BJ. La concentration des cellules devraient être d&#39;environ 5 x 10 4 cellules / puits. Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Remplacer moyenne 24 heures après le réensemencement avec des humains ES / IPS milieu de culture cellulaire (400 ml DMEM/F12 supplémenté avec 100 ml de sérum de remplacement Knockout, 5 ml 10 mM non acides aminés essentiels, 5 ml de 200 mM de L-glutamine, 0,9 ml 55 mM β-mercaptoéthanol et 20 ng / ml de bFGF humain recombinant). Changer moyenne toutes les 24 heures pendant 7 jours. </li><li> Après sept jours, le changement moyen du MEF milieu conditionné (voir section 2). </li></ol> 2. Préparation du MEF milieu conditionné <ol><li> Graine FC-1 des cellules nourricières du MEF à 2 x 10 5 cellules / puits sur une plaque à 6 puits dans 2 ml de milieu de croissance du MEF. Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Changement moyen humaines ES / IPS milieu de culture cellulaire. Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Recueillir le surnageant toutes les 24 heures pendant quatre jours. Filtrer le surnageant à travers un filtre de 0,22 um. </li><li> Supplément surnageant avec 50 ng / ml de bFGF. </li></ol> 3. Sélection Colony iPS et repiquage <ol><li> Sélectionnez une colonie ES-like et réensemencer en milieu de culture humaine ES / IPS complété avec 10 pM de Stemolecule Y27632 sur des boîtes de culture de cellules pré-ensemencés avec des CF-1 des cellules nourricières du MEF. Incuber une nuit à 37 ° C et CO 2 5%. </li><li> Changer le milieu de culture toutes les 24 heures pour les sept premiers jours (sans complétant avec Stemolecule Y27632). Après sept jours, le changement moyen du MEF milieu conditionné (voir section 2 pour la préparation). Nous avons continué repiquage des cellules jusqu&#39;à ce qu&#39;elles montrent la morphologie typique ES humaines. </li></ol> 4. Examen des marqueurs immunocytochimiques pluripotence <ol><li> Lavez délicatement les cellules trois fois avec PBS. </li><li> Fixer les cellules avec 500 pi de fixateur pendant 20 minutes à température ambiante. </li><li> Lavez délicatement les cellules trois fois avec PBS. </li><li> Bloc de fixation non spécifique avec 500 ul du tampon de blocage pendant une heure à température ambiante. </li><li> Incuber les cellules avec 250 pi de l&#39;anticorps primaire spécifique nuit à 4 ° C (nous avons utilisé TRA-1-81, TRA-1-60, SSEA-4, SSEA-3, Oct4, Sox2, Nanog et Lin28 au 1:100 dilutions). </li><li> Lavez délicatement les cellules trois fois avec PBS. </li><li> Incuber les cellules avec 250 pi d&#39;anticorps secondaire pendant 1 heure à température ambiante, en gardant loin de la lumière (nous avons utilisé chèvre anti-souris conjugué IgM Cy3, de chèvre anti-IgG de souris conjugué Cy3, chèvre anti-rat IgM Cy3 conjugué et de chèvre anti- Lapin Cy3 conjugué au 1:300 dilutions). </li><li> Lavez délicatement les cellules trois fois avec PBS. </li><li> Ajouter DAPI (concentration finale 1 ug / ml) pour le dernier lavage et incuber 5 minutes à visualiser les noyaux. </li><li> Analyser les cellules sous grossissement. </li></ol> Partie 5: Résultats Représentant 1. Résultats Morphologie: L&#39;homme le prépuce fibroblastes (BJ), les cellules ont été co-transduites avec Oct4, Sox2, Nanog, et Lin28. Les modifications morphologiques ont été observées dès le jour 4 post-transduction, et les amas de cellules sont devenues plus serrées au jour 17 (figure 1). Des colonies ont été cueillis à la main 25 jours et cultivés sur FC-1 des cellules nourricières du MEF. Afin de faciliter la formation iPS colonie cellulaire après reprogrammation, nous avons utilisé Stemolecule Y27632, Un inhibiteur de ROCK, pour l&#39;ensemencement initial nuit lors de chaque passage. colonies de cellules iPS avec une bonne morphologie ont été observées après trois cycles séquentiels de la colonie de la cueillette et repiquage. 2. Expression de marqueurs pluripotence: Afin de mieux caractériser les cellules iPS colonies isolées, nous avons examiné pour la présence de marqueurs communs pluripotence exprimé dans les cellules ES. Les colonies présentaient une forte phosphatase alcaline (AP) activité (figure 2). Par ailleurs, l&#39;immunocytochimie (ICC) a été réalisée sur les colonies de cellules iPS avec un panel d&#39;anticorps marqueurs spécifiques pluripotence, y compris les marqueurs de surface TRA-1-81, TRA-1-60, SSEA-4 et SSEA-3 ainsi que des marqueurs nucléaires Oct4, Sox2 et Nanog. Les colonies isolées iPS ont été positifs pour tous les marqueurs (figure 3). Les résultats de la CPI montrent que les cellules iPS exposées du motif de la pluripotence appropriée l&#39;expression du marqueur, ce qui démontre que ces cellules iPS ressemblent indifférencié des cellules ES humaines. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Figure 1" /> Figure 1: Les modifications morphologiques des cellules transduites BJ. Images en champ clair d&#39;une colonie de cellules iPS typiques formé en (A) 4 jours et (B) 17 jours après la transduction. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Figure 2" /> Figure 2: Activité de l&#39;AP reprogrammé les cellules BJ. Trois différentes colonies colorées à Stemgent Kit Phosphatase Alcaline coloration. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Figure 3" /> Figure 3: Les cellules iPS humaines expriment des niveaux élevés des marqueurs de cellules ES suivantes spécifiques: les marqueurs de surface TRA-1-81, TRA-1-60, SSEA-4 et SSEA-3, et des marqueurs nucléaires 4 oct. Nanog et Sox2.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

Ces résultats démontrent que l&#39;homme Stemgent Set Lentivirus TF peut être utilisé pour générer efficacement des colonies iPS en induisant l&#39;expression ectopique de transduction des facteurs de transcription dans des fibroblastes humains. Lors de la conception des expériences de reprogrammation, plusieurs variables doivent être considérés afin d&#39;optimiser l&#39;efficacité de la reprogrammation. Premièrement, le virus de l&#39;actif à objectif de ratio (multiplicité d&#39;infection MOI) peuvent avoir besoin d&#39;être modifiés durant l&#39;étape de la transduction du primaire à atteindre une efficacité optimale de transduction. Deuxièmement, la condition de croissance des cellules cibles peuvent avoir un impact reprogrammation. Les cellules saines et proliférative se prêtent davantage à une reprogrammation. Troisièmement, lors de la modification du protocole pour le nombre de cellules différentes, il est recommandé que le nombre de cellules cibles être ajustée proportionnellement à la surface de la boîte de culture. Enfin, les inhibiteurs de ROCK application tels que Y27632 devrait être considérée pour aider à assurer reprogrammation de succès que des études récentes ont démontré son utilité dans l&#39;amélioration de la survie des colonies hES 4,5.

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<td>Human TF Lentivirus Set</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

En 2006, Yamanaka et ses collègues ont d&#39;abord démontré que des rétrovirus médiation de livraison et d&#39;expression de Oct4, Sox2, c-Myc et Klf4 est capable d&#39;induire l&#39;état pluripotent dans des fibroblastes de souris. 1 Le même groupe a également signalé la reprogrammation réussie de cellules somatiques humaines dans les souches pluripotentes induites (iPS) en utilisant des versions humaines des facteurs de transcription mêmes livrés par des vecteurs rétroviraux. 2 En outre, James Thomson et al. signalé que les lentivirus médiée par la co-expression d&#39;un autre ensemble de facteurs (Oct4, Sox2, Nanog et Lin28 ) a été capable de reprogrammer des cellules somatiques humaines en cellules iPS. 3 Les cellules iPS sont similaires aux cellules ES dans la morphologie, la prolifération et la capacité de se différencier en tous types de tissus du corps. Cellules iPS humaines ont un net avantage sur les cellules ES comme ils présentent des propriétés clés des cellules ES sans le dilemme éthique de la destruction d&#39;embryons. La génération de cellules iPS spécifiques au patient contourne un barrage important de personnaliser les thérapies en médecine régénérative, en éliminant le risque de rejet immunitaire de la non-autologues cellules transplantées. Ici nous démontrons le protocole pour la reprogrammation des cellules de fibroblastes humains en utilisant les Stemgent Set homme Lentivirus TF. Nous montrons aussi que les cellules reprogrammées avec cet ensemble commencent à montrer des iPS morphologie quatre jours après la transduction. Utilisation de la Y27632 Stemolecule, nous avons sélectionné pour les cellules iPS et observé la morphologie correcte après trois cycles séquentiels de la colonie de la cueillette et repiquage. Nous démontrons également que, après reprogrammation des cellules affiché le marqueur AP pluripotence, les marqueurs de surface TRA-1-81, TRA-1-60, SSEA-4, et SSEA-3, et des marqueurs nucléaires Oct4, Sox2 et Nanog.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Nous démontrons le protocole pour la génération de cellules souches pluripotentes induites à partir des cellules somatiques humaines en utilisant des lentivirus médiée par la livraison des facteurs humains Oct4, Sox2, Nanog, et Lin28. Pluripotence a été confirmée par la morphologie et la présence de cellules souches embryonnaires (ES) de cellules de marqueurs spécifiques.

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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Unit 1

Embryonic and Induced Pluripotent Stem Cells

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29.7K vues.  Stemgent. Nous démontrons le protocole pour la génération de cellules souches pluripotentes induites à partir des cellules somatiques humaines en utilisant des lentivirus médiée par la livraison des facteurs humains Oct4, Sox2, Nanog, et Lin28. Pluripotence a été confirmée par la morphologie et la présence de cellules souches embryonnaires (ES) de cellules de marqueurs spécifiques.

Vidéo: Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Culture et entretien des cellules souches embryonnaires humaines

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Génération de cellules souches pluripotentes induites par la reprogrammation des fibroblastes embryonnaires de souris avec un facteur de transcription Quatre, doxycycline inductible système de transduction lentiviraux

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congélation et de décongélation cellules souches embryonnaires humaines

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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 ...

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 ...