1. Riprogrammazione <ol><li> BJ cellule seme ad una densità di 1 x 10 5 cellule per pozzetto di un 6-pozzetti. Coltivare le cellule in 2 ml di mezzo di crescita (450 ml Emem integrata con 50 ml di FBS ES qualificati, 5 ml di 10 mM di aminoacidi non essenziali, 5 ml di penicillina / streptomicina, 5 ml di 200 mM di L-glutamina e 0,9 ml di 55 mM β -mercaptoetanolo) overnight a 37 ° C e 5% di CO 2. </li><li> Lo stesso giorno, iniziare a preparare piatti alimentatore MEF con l&#39;aggiunta di 0,1% di gelatina diluita in acqua per un piatto ben 6 e incubazione overnight a 37 ° C e CO 5% 2. </li><li> Dopo l&#39;incubazione, rimuovere media dalle cellule BJ e aggiungere 2 ml di terreno di coltura integrata con 6 mg / ml di polibrene, 500 microlitri hOct4-lentivirus, 50 microlitri hSox2-lentivirus, 50 microlitri hNanog-lentivirus e 50 microlitri hLin28-lentivirus in ciascun pozzetto . Agitare delicatamente la piastra per assicurare uno strato uniforme del mezzo. Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Lo stesso giorno, togliere 1 fiala di CF-1 le cellule MEF da azoto liquido e disgelo. Rimuovere il liquido dai pozzetti rivestiti di gelatina (vedi punto 1.2) e aggiungere le cellule MEF alimentatore ad una densità di 0,2 x 10 5 cellule / pozzetto. Il volume totale per bene dovrebbe essere 2 ml di cellule in terreno di crescita MEF (450 ml di DMEM integrato con 50 FBS ml e 5 ml di aminoacidi non essenziali). </li><li> Il giorno dopo, staccare le cellule BJ con il 0,05% tripsina / EDTA e centrifugare a 200 xg per 5 minuti. Aspirare il terreno e risospendere in mezzo di crescita. Togliere dalla piastra medio alimentatore MEF (vedi punto 1.4) e aggiungere 2 ml / pozzetto della sospensione cellulare BJ. La concentrazione di cellule dovrebbe essere di circa 5 x 10 4 cellule / pozzetto. Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Sostituire media 24 ore dopo la risemina con umane ES / IPS mezzo di coltura cellulare (400 ml DMEM/F12 integrato con 100 ml di sostituzione Knockout siero, 5 ml di 10 mM di aminoacidi non essenziali, 5 ml di 200 mM L-glutammina, 0,9 ml 55 mM β-mercaptoetanolo e 20 ng / ml bFGF umano ricombinante). Cambia media ogni 24 ore per 7 giorni. </li><li> Dopo sette giorni, cambio medio a medio MEF condizionata (vedi punto 2). </li></ol> 2. Preparazione del mezzo MEF condizionata <ol><li> Seme CF-1 le cellule MEF alimentatore con 2 x 10 5 cellule / pozzetto su un piatto ben 6 in 2 ml di mezzo di crescita MEF. Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Cambia medio umane ES / IPS mezzo di coltura cellulare. Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Raccogliere il supernatante ogni 24 ore per quattro giorni. Filtrare il surnatante attraverso un filtro a 0,22 micron. </li><li> Supplemento surnatante con 50 ng / ml di bFGF. </li></ol> 3. iPS selezione Colony e Passaging <ol><li> Selezionare un ES-come colonia e ri-seme umano ES / IPS mezzo di coltura integrata con 10 mM di Stemolecule Y27632 sui piatti di coltura cellulare pre-seminati con cellule MEF CF-1 alimentatore. Incubare per una notte a 37 ° C e 5% di CO 2. </li><li> Cambiare la cultura media ogni 24 ore per i primi sette giorni (senza l&#39;integrazione con Stemolecule Y27632). Dopo sette giorni, cambio medio a medio MEF condizionata (vedi sezione 2 per la preparazione). Abbiamo continuato passaging le cellule fino a quando non hanno mostrato tipica morfologia umana ES. </li></ol> 4. Esame immunocitochimica di marcatori pluripotenza <ol><li> Lavare le cellule delicatamente per tre volte con PBS. </li><li> Fissare le cellule con 500 ml di fissativo per 20 minuti a temperatura ambiente. </li><li> Lavare le cellule delicatamente per tre volte con PBS. </li><li> Blocca legame non specifico con 500 microlitri di buffer bloccando per un&#39;ora a temperatura ambiente. </li><li> Incubare le cellule con 250 microlitri di anticorpo specifico primario overnight a 4 ° C (abbiamo usato TRA-1-81, TRA-1-60, SSE-4, SSE-3, Oct4, Sox2, e Nanog Lin28 a 1:100 diluizioni). </li><li> Lavare le cellule delicatamente per tre volte con PBS. </li><li> Incubare le cellule con 250 microlitri di anticorpo secondario per 1 ora a temperatura ambiente, mantenendo al riparo dalla luce (abbiamo usato capra anti-topo coniugato IgM Cy3, capra anti-topo IgG Cy3 coniugato, capra anti-topo coniugato IgM Cy3 e di capra anti- Cy3 coniglio coniugato a diluizioni 1:300). </li><li> Lavare le cellule delicatamente per tre volte con PBS. </li><li> Aggiungi DAPI (concentrazione finale 1 mg / ml) per l&#39;ultimo lavaggio e incubare 5 minuti per visualizzare nuclei. </li><li> Analizzare le cellule sotto ingrandimento. </li></ol> Parte 5: Risultati Rappresentante 1. Morfologia dei risultati: Prepuzio fibroblasti umani (BJ), le cellule sono state co-trasdotte con Oct4, Sox2, Nanog, e Lin28. Cambiamenti morfologici sono stati osservati già dal 4 ° giorno post-trasduzione, e il cluster di cellule è diventato più fitto al giorno 17 (Figura 1). Colonie sono state raccolte manualmente al giorno 25 e coltivati ​​su cellule CF-1 alimentatore MEF. Per facilitare la formazione delle cellule iPS colonia dopo la riprogrammazione, abbiamo usato Stemolecule Y27632, Un inibitore ROCK, per la semina iniziale durante la notte durante ogni passaggio. colonie di cellule iPS con buona morfologia sono stati osservati dopo tre gare in sequenza di colonia picking e passaging. 2. Espressione di marcatori pluripotenza: Per caratterizzare ulteriormente le colonie isolate cellule iPS, abbiamo cercato la presenza di marcatori di pluripotenza comuni espressi nelle cellule ES. Le colonie esposte forte fosfatasi alcalina (AP) attività (Figura 2). Inoltre, immunocitochimica (ICC) è stata effettuata su colonie di cellule iPS la con un pannello di anticorpi specifici marker pluripotenza, tra cui marcatori di superficie TRA-1-81, TRA-1-60, SSE-4 e SSE-3 così come marcatori nucleari Oct4, Sox2 e Nanog. Le colonie isolate iPS sono stati positivi per tutti gli indicatori (Figura 3). I risultati ICC mostrano che le cellule iPS esposto l&#39;appropriato marcatore di pluripotenza pattern di espressione, a dimostrazione che queste cellule iPS assomigliano molto indifferenziate cellule staminali embrionali umane. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="Figura 1" /> Figura 1: i cambiamenti morfologici delle cellule trasdotte BJ. Immagini in campo chiaro di una tipica colonia di cellule iPS formata in (A) 4 giorni e (B) 17 giorni dopo trasduzione. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="Figura 2" /> Figura 2: attività di AP riprogrammato cellule BJ. Tre diverse colonie colorate con Stemgent Kit di colorazione fosfatasi alcalina. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="Figura 3" /> Figura 3: le cellule umane iPS esprimono alti livelli dei marker delle cellule staminali seguenti specifiche: marcatori di superficie TRA-1-81, TRA-1-60, SSE-4 e SSE-3, e marcatori nucleari 4 ottobre, Sox2 e Nanog.

<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>

Gli autori di questo articolo sono impiegati da Stemgent che produce reagenti e strumenti utilizzati in questo articolo.

Questi risultati dimostrano che la Stemgent umani Set Lentivirus TF può essere usato per generare in modo efficiente le colonie iPS inducendo l&#39;espressione ectopica di trasduzione fattori di trascrizione in fibroblasti umani. Nel progettare gli esperimenti di riprogrammazione, diverse variabili dovrebbero essere considerate per ottimizzare l&#39;efficienza della riprogrammazione. In primo luogo, il virus attivo rispetto al traguardo rapporto (molteplicità di infezione MOI) può avere bisogno di essere modificati durante la fase di trasduzione primario di raggiungere la massima efficienza di trasduzione. In secondo luogo, la condizione di crescita delle cellule bersaglio può avere un impatto riprogrammazione. Cellule sane e proliferativi sono più suscettibili di riprogrammazione. In terzo luogo, quando si modifica il protocollo per numero di cellule differenti, si raccomanda che il numero di cellule bersaglio essere adeguato in proporzione alla superficie del piatto cultura. Infine, gli inibitori ROCK applicando come Y27632 deve essere considerato per garantire la riprogrammazione di successo come studi recenti hanno dimostrato la propria utilità nel migliorare la sopravvivenza colonia hES. 4,5

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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

Nel 2006 Yamanaka e colleghi hanno dimostrato che i retrovirus-mediata consegna ed espressione di Oct4, Sox2, c-Myc e Klf4 è in grado di indurre lo stato pluripotente in fibroblasti di topo. 1 Lo stesso gruppo ha anche riferito del successo riprogrammazione di cellule somatiche umane in staminali pluripotenti indotte (iPS), le cellule umane usando versioni dei fattori di trascrizione stessa forniti da vettori retrovirali. 2 Inoltre, James Thomson et al. riferito che il lentivirus-mediata co-espressione di un altro insieme di fattori (Oct4, Sox2, e Nanog Lin28 ) è stato in grado di riprogrammare cellule somatiche umane in cellule iPS 3. cellule iPS sono simili alle cellule staminali embrionali nella morfologia, la proliferazione e la capacità di differenziarsi in tutti i tipi di tessuto del corpo. Cellule umane iPS hanno un netto vantaggio rispetto cellule ES in quanto mostrano proprietà fondamentali delle cellule ES senza il dilemma etico della distruzione degli embrioni. La generazione di paziente-specifiche cellule iPS eludere un posto di blocco importante di terapie personalizzate medicina rigenerativa, eliminando il rischio di rigetto immunitario delle cellule trapiantate non autologo. Qui mostriamo il protocollo per la riprogrammazione delle cellule di fibroblasti umani utilizzando il Stemgent umani Set Lentivirus TF. Mostriamo anche che le cellule riprogrammate con questa serie iniziano a mostrare iPS morfologia quattro giorni dopo la trasduzione. Utilizzando il Stemolecule Y27632, abbiamo selezionato per le cellule iPS e osservato corretta morfologia dopo tre round di colonia picking e passaging. Abbiamo anche dimostrato che, dopo la riprogrammazione delle cellule visualizzata la pluripotenza marcatore AP, marcatori di superficie TRA-1-81, TRA-1-60, SSE-4, e SSE-3, e marcatori nucleari Oct4, Sox2 e 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

Dimostriamo il protocollo per la generazione di cellule staminali pluripotenti indotte da cellule somatiche umane utilizzando lentivirus-mediata consegna dei fattori umani Oct4, Sox2, Nanog, e Lin28. Pluripotenza è stata confermata dalla morfologia e la presenza di staminali embrionali (ES) cellulo-specifici marcatori.

Generazione di cellule staminali pluripotenti indotte da fibroblasti umani Riprogrammare con il Stemgent Lentivirus umani TF 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 Views.  Stemgent. Dimostriamo il protocollo per la generazione di cellule staminali pluripotenti indotte da cellule somatiche umane utilizzando lentivirus-mediata consegna dei fattori umani Oct4, Sox2, Nanog, e Lin28. Pluripotenza è stata confermata dalla morfologia e la presenza di staminali embrionali (ES) cellulo-specifici marcatori.

Video: 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.

Cultura e manutenzione di cellule staminali embrionali umane

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.

Generazione di cellule staminali pluripotenti indotte da Riprogrammazione fibroblasti embrionali di topo con un fattore quattro trascrizione, Doxiciclina inducibile lentivirali sistema di trasduzione

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.

Congelamento e scongelamento cellule staminali embrionali umane

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.

Trasfettando e Nucleofecting umani cellule staminali pluripotenti indotte

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.

Selezione e isolamento di colonie di cellule pluripotenti indotte umane staminali riprogrammato da fibroblasti adulti

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.

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con 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.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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.

Generazione di cellule pluripotenti indotte umane staminali da sangue periferico mediante il STEMCCA lentivirale Vector

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.

Generazione di cardiomiociti umani: Un protocollo di differenziazione da alimentatore-Free umani cellule staminali pluripotenti indotte

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.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con 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 ...