Lavoro descritto in questo manoscritto è stato reso possibile da un finanziamento della California Institute for Regenerative Medicine (CIRM) per Core cellule staminali UCR.

Il nostro protocollo inizia con un metodo di adattarsi a culture umane iPSCs alimentatore-libera, seguito da protocolli per trasfezione iPSCs umani utilizzando GeneJuice (EMD) e nucleofection di iPSCs umani utilizzando un dispositivo di Amaxa nuclefector. Nota: Le seguenti procedure sono eseguite in una cappa a flusso laminare sterile. Tutti i mezzi e le soluzioni sono equilibrati a 37 ° C oa temperatura ambiente prima di iniziare se non diversamente specificato. 1. Stabilire iPSCs umani sul sistema privo di alimentatore IPSCs umani precedentemente detenute nelle cellule di alimentazione possono essere divisi, trasferiti su Geltrex rivestite piatto e mantenuta per due passaggi prima di alimentazione priva di trasfezione. <ol><li> Scongelare Geltrex notte a 4 ° C. Per preparare rivestimento Geltrex, diluire scongelato Geltrex 1:50 in DMEM freddo. Mescolare delicatamente le soluzioni. </li></ol> Nota: Geltrex, come Matrigel è una forma solubile di matr membrana basaleix purificati da murino Engelbreth-Holm-Swarm (EHS) cellule tumorali. In alternativa, Matrigel può essere utilizzato come matrice extracellulare stabilire culture umane alimentatore-libere IPSC. <ol start="2"><li> Coprire l&#39;intera superficie dei pozzetti di coltura con Geltrex soluzione (1 ml per 35 mm oltre). Cappotto pozzetti con Geltrex a 37 ° C incubatore per 1 ora. </li><li> Per iPSCs passaggio umano, aggiungere 1 ml di accutase per pozzetto e incubare a 37 ° C per 1 minuto fino a quando la maggior parte delle cellule iniziano a staccarsi. </li></ol> Per iPSCs passaggio umano, aggiungere 1 ml di accutase per pozzetto e incubare a 37 ° C per 1 minuto fino a quando la maggior parte delle cellule iniziano a staccarsi. <ol start="4"><li> Aggiungere 10-15 perle di vetro per le cellule e mescolare delicatamente la piastra. Aggiungere 2 ml di DMEM KnockOut / F12 e delicatamente triturare. Trasferire sospensione cellulare in un tubo da centrifuga da 10 ml. </li><li> Centrifugare le cellule a 800 rpm per 3 minuti a temperatura ambiente. </li><li> Aspirare il surnatante dal tubo, lasciando intatta umana iPSC pellet. Delicatamente scatti la provetta perdisperdere pellet. </li><li> Rimuovere Geltrex dal pozzo rivestito. </li><li> Risospendere delicatamente umano iPSC pellet in un volume adeguato di STEMPRO. Distribuire tra i pozzetti di alimentatori, a seconda del tasso di proliferazione). IPSCs umani possono essere diversi passaggi in un rapporto di divisione compreso tra 1:2 e 1:6. </li><li> Con cautela in luogo del 5% CO 2 incubatore, agitare la piastra con attenzione per garantire una distribuzione uniforme delle cellule in tutto il pozzetto. </li><li> Le cellule di alimentazione tutti i giorni fino cellule sono pronte per essere dividere di nuovo (quando le cellule raggiungono l&#39;80% di confluenza). </li><li> IPSCs passaggio umano sul nuovo pozzo Geltrex rivestita passo (1,3-1,9) in un rapporto di divisione di 1:2. Piccole colonie dovrebbero essere formati e distribuiti uniformemente su Geltrex rivestita bene prima trasfezione. </li></ol> 2. La trasfezione di iPSCs umani con GeneJuice (Cellule coltivate in piastre da 6 pozzetti) deve essere di circa 40 -50% confluenti nel giorno della trasfezione per ottenere un rendimento ottimale trasfezione. Ènon occorre modificare il mezzo cella fino al giorno successivo. <ol><li> Preparare 100 KO-DMEM/F12 ul in un tubo sterile 1,5 ml Eppendorf. Aggiungere 27 microlitri di reagente di trasfezione GeneJuice. Mescolare bene. Incubare a temperatura ambiente per 5 minuti. </li><li> Aggiungere 4 mg DNA plasmidico. Incubare la provetta a temperatura ambiente per 15 minuti. La scelta del plasmide è critico per l&#39;efficienza di trasfezione ottimale. Usiamo un plasmide con una proteina maggiore fluorescenza verde (EGFP) guidato da CAG promotore 5 (pCAG-eGFP). CAG promotore è un promotore forte che è trascrizionalmente attivo in iPSCs umani e quindi può essere usato per guidare l&#39;espressione del transgene in queste cellule. Nelle nostre mani, linearizzazione del plasmide non sembra influenzare l&#39;efficienza di trasfezione. </li><li> Aggiungi GeneJuice-DNA miscela alle cellule e agitare la piastra. Centrifugare la piastra a 1200 rpm per 5 minuti (metodo &#39;spinoculation&#39;) per aumentare il contatto della miscela di trasfezione con iPSCs umani sul pozzo. </li><li> Incubare le cellule a 37 &amp;deg; C per una notte. </li><li> Monitorare l&#39;efficienza di trasfezione il giorno successivo. </li><li> Per la trasfezione stabile, modificare media il giorno successivo con STEMPRO fresco. Aggiungere appropriata selezione antibiotica dopo 24 - 48 ore. </li></ol> 3. Nucleofection di iPSCs umani IPSCs umani cresciuti su alimentatori o Geltrex può essere utilizzato per nucleofection. Tuttavia, si consiglia vivamente di replating nucleofected iPSCs umani su fibroblasti embrionali di topo (MEF) o fibroblasti prepuzio umano (HFF) alimentatori per garantire alta vitalità cellulare e di recupero. Almeno 2 milioni di cellule devono essere utilizzati per ottenere la sopravvivenza delle cellule superiore a seguito nucleofection. <ol><li> Preparare alimentatore cellule mezzi condizionati (CM) incubando umana KnockOut iPSC sierici di ricambio (KOSR) supporto con cellule di alimentazione durante la notte. Raccogliere CM ogni 24 ore. </li></ol> Nota: Qualsiasi ceppo di topi utilizzati per la preparazione di strati alimentatore (come BLK6, CF1 e MF1) è adatto per essere utilizzato for rendendo CM. <ol start="2"><li> Il giorno della nucleofection, pre-trattare iPSCs umani con 10 pM ROCK inibitore per almeno 1 ora. </li><li> Preparare 82 ml di soluzione umana Nucleofector cellule staminali in una sterile 1,5 ml tubo eppendorf. Aggiungere 18 microlitri supplemento 1. Mescolare bene. Incubare la soluzione a 37 ° C per 5 minuti. </li><li> Preriscaldare alimentatore cella condizionata media (CM) e 0,25% tripsina / EDTA a 37 ° C. Sotto il cofano, prelabel uno sterile da 15 ml tubo conico. </li><li> Rimuovere con cautela i supporti dal cultura umana iPSC da nucleofected. Lavare delicatamente le cellule con 2 ml di soluzione di fosfato 1X tamponata (PBS) per pozzetto. Aspirare il PBS. Aggiungere 1 ml di 0,25% tripsina per pozzetto. Incubare le cellule a 37 ° C per 3 minuti. </li><li> Delicatamente triturare le cellule con 1000 ml di punta della pipetta. Lavare il fondo del pozzo per assicurarsi che tutti iPSCs umani sono completamente staccato. Trasferire sospensione cellulare alla provetta etichettata 15 ml. </li></ol> Nota: tripsinizzazione in singolo cells va rigorosamente evitato. Le cellule devono essere sloggiato in piccoli ciuffi di cellule composto da circa triplette di cellule. Piccoli ciuffi di cellule (cellule triple) sarà dissociato in singole cellule durante la successiva manipolazione delle cellule. <ol start="7"><li> Aggiungere 9 ml supporti MEF per inattivare la tripsina. Spin le cellule a 800 rpm per 3 minuti. Aspirare accuratamente il supernatante e lasciare il pellet di cellule intatte. </li><li> Risospendere le cellule in preriscaldato 100 ul di soluzione di cellule staminali umane Nucleofector dal punto 3.3. </li><li> Trasferire le cellule a una cuvetta Nucleofector utilizzando un ml 1 puntale. </li><li> Aggiungere 4 pg di DNA plasmidico in sospensione di cellule nella cuvetta. Mescolare cellule e il DNA agitando delicatamente. Toccare la cuvetta due volte sulla superficie del cofano. </li><li> Inserire la cuvetta nel porta Nucleofector. Utilizzare programmi B-016. Cellule Nucleofect premendo il pulsante X. </li></ol> &lt; <ok> &gt; Verrà visualizzato quando il processo nucleofection è completed (di solito richiede solo 1-5 secondi). L&#39;uso di altri programmi nucleofection (A-023, A-033 e U-023) prodotto meno del 10% di cellule positive eGFP da iPSCs umane. </ok><ol start="12"><li> Recuperare le cellule nucleofected dalla cuvetta utilizzando l&#39;apposita pipetta Pasteur di plastica. Recuperare le cellule da risospensione in CM preriscaldato e inibitore ROCK in una sterile 1,5 ml tubo eppendorf. Incubare le cellule a 37 ° C per 10 minuti per far recuperare cellule. </li><li> Cellule di trasferimento goccia a goccia su strati alimentazione con 1 ml punta della pipetta. Incubare le cellule a 37 ° C per una notte. </li><li> Monitorare l&#39;efficienza di trasfezione il giorno successivo. Per derivare cloni stabili di transgenici iPSCs umani che esprimono stabilmente transgene, cambiare mezzo il giorno dopo con CM e inibitore ROCK. Aggiungere appropriata selezione antibiotica dopo 24 - 48 ore. </li></ol> 4. Rappresentante dei risultati: <img alt="Figura 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <strong&gt; Figura 1. Microfotografie di Riv1 iPSCs umane trasfettate con pCAG-eGFP. (A) eGFP-esprimenti Riv1 cellule trasfettate con il GeneJuice Geltrex 12 ore dopo la trasfezione. Le colonie di Riv1 iPSCs umani placcato e formato su linee seguenti nucleofection con B-016 (B) e A-023 (C) del programma. (D) che esprimono stabilmente eGFP-colonie umane IPSC con espressione eGFP ubiquitaria derivato da GeneJuice. (E) stabilmente transfettate Riv1 iPSCs umani conservati espressione costitutiva eGFP durante il differenziamento del corpo embryoid.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, 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=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Il nostro risultato protocolli in tecniche semplici, robusti e altamente riproducibile per introdurre transgeni in iPSCs umani senza effetti tossici di rilievo e la morte cellulare. IPSCs umani devono essere diversi passaggi in piccoli grumi di cellule (cellule 5-10) e piastrati su Geltrex ad alta densità (1:2) per garantire la massima efficienza di transfezione in numerose piccole colonie. Per le linee IPSC umani che sono più inclini a differenziazione e morte cellulare, maggior numero di iPSCs umani (fino a 4 x 10 6 cellule) deve essere utilizzato per un esperimento nucleofection unico. Saggio di trasfezione transiente genera un gran numero di transgenici che esprimono iPSCs umani entro 1 giorno. Trasfettate stabilmente cloni IPSC di solito appaiono entro 7 giorni, e queste colonie transgenici dovrebbe essere pronto per essere raccolto entro tre settimane. L&#39;uso di promotore descritto qui CAG assicura espressione ubiquitaria di reporter eGFP. In tali miglioramenti, i nostri protocolli possono essere inseriti in altre applicazioni, tra cui sovraespressione, cinduzione talune condizioni, derivazione di lineage-specifiche linee di giornalista, shRNA o knockdown siRNA, ricombinazione gene targeting e omologhi.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 0,25% tripsina con EDTA </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2-Mercaptoethanol </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3 biglie di vetro millimetri </td><td> Fisher Scientific </td><td> 11-312A </td><td> Perle di vetro deve essere lavato con acido cloridrico (HCl) overnight, risciacquato con idrossido di sodio (NaOH) e acqua distillata e sterilizzato in autoclave prima dell&#39;uso. </td></tr><tr><td> Accutase reagente dissociazione cellulare </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> Base del fattore di crescita dei fibroblasti (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (glucosio) </td&gt; <td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> Siero fetale di vitello </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> Fattore di crescita ridotta </td></tr><tr><td> GeneJuice trasfezione reagente </td><td> EMD Biosciences </td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I (100X) </td><td> Invitrogen </td><td> 35050061 </td><td> L-glutammina (Invitrogen, 25.030.081) può anche essere utilizzato. </td></tr><tr><td> Umana iPSC KOSR supporti </td><td></td><td></td><td> 500 ml umano ES media consiste di 390 KnockOut ml DMEM / F12, 100 ml di siero KnockOut Replacer, 5ml Glutamax-I (100X), 5ml NEAA (100X), 500 pl 2-mercaptoetanolo (55mm) e completata con 10 ng / ml bFGF . </td></tr><tr><td> KnockOut DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td>KnockOut sostituzione Siero (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> Fibroblasti embrionali di topo supporti </td><td></td><td></td><td> MEF supporto consiste di 90% FBS, NEAA 1X, 1X Glutamax-I e sodio piruvato in DMEM 1X (glucosio) </td></tr><tr><td> Non aminoacido essenziale, NEAA? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> Cellule staminali umane soluzione Nucleofector 1 con supplemento </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> Soluzione tampone fosfato senza Ca 2 + e Mg 2 + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> Piruvato di sodio (100X) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO media kit </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ml STEMPRO media consiste di 454 ml di KnockOut DMEM / F12, 10 ml STEMPRO privo di siero supplemento di crescita (50X), 5ml Glutamax-I (100X), 5ml NEAA (100X), 36 ml di BSA (albumina sierica bovina, 25%), 909 microlitri 2-mercaptoetanolo (55mm ), integrato con 10 ng / ml bFGF. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

La modificazione genetica continua ad essere uno strumento essenziale per studiare biologia delle cellule staminali e, indicando i potenziali applicazioni cliniche delle cellule staminali embrionali umane (hESC) 1. Mentre i miglioramenti in diversi metodi di consegna del gene sono state descritte 2-9, trasfezione resta un processo per hESC capricciosa, e non è ancora stata riportata in cellule staminali pluripotenti indotte umane (iPSCs). In questo video, dimostriamo come il nostro laboratorio di routine e transfects nucleofects iPSCs umani utilizzando plasmide con un avanzato proteina verde fluorescente (eGFP) giornalista. IPSCs umani sono adattati e mantenuti come alimentatore-libere culture per eliminare la possibilità di trasfezione cellulare alimentatore e consentire efficiente selezione di cloni stabili transgenici IPSC dopo trasfezione. Per nucleofection, iPSCs umani sono pre-trattati con inibitori ROCK 11, tripsinizzate in piccoli ciuffi di cellule, nucleofected e saltuariamente su linee di alimentazione in cella climatizzatamezzo per migliorare il recupero delle cellule. Transgene-esprimenti iPSCs umani può essere ottenuta dopo 6 ore. Selezione antibiotica è applicato dopo 24 ore e stabili linee transgeniche sembrano entro 1 settimana. Il nostro protocollo è robusto e riproducibile per umani linee IPSC senza alterare pluripotenza di queste cellule.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Nonostante i recenti progressi nella modificazione genetica, trasfezione di cellule staminali embrionali umane (hESC) rimane un processo capriccioso. A nostra conoscenza, metodi sistematici ed efficiente per trasfettare umani cellule staminali pluripotenti indotte (iPSCs) non sono stati segnalati. Qui, descriviamo i protocolli affidabili per trasfettare in modo efficiente e nucleofect iPSCs umani.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.4K Views.  University of California Riverside. Nonostante i recenti progressi nella modificazione genetica, trasfezione di cellule staminali embrionali umane (hESC) rimane un processo capriccioso. A nostra conoscenza, metodi sistematici ed efficiente per trasfettare umani cellule staminali pluripotenti indotte (iPSCs) non sono stati segnalati. Qui, descriviamo i protocolli affidabili per trasfettare in modo efficiente e nucleofect iPSCs umani.

Video: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Trasfezione delle cellule staminali neuronali con il Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Ricerca

Biologia

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.

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparazione di cellule di fibroblasti embrionali di topo adatto per colture di cellule pluripotenti umane embrionali e staminali indotta

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

Riprogrammazione episomiale dei fibroblasti dermici per generare cellule staminali pluripotenti indotte dall'uomo (hiPSCs)

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.