Questi studi sono stati finanziati in parte dal NIH UO1HL107443-01 Premio per GJM e GM.

1. Isolamento ed espansione delle cellule mononucleari del sangue periferico (PBMC) GIORNO 0 <ol><li> Disegnare 4 ml di sangue periferico in una provetta BD Preparazione Vacutainer CPT cellulare con citrato di sodio. La provetta 8 a 10 volte e centrifugare a 1800 xg per 30 min a temperatura ambiente. Idealmente, questo passo dovrebbe essere fatto entro 2 ore dal prelievo. </li><li> Raccogliere le cellule mononucleate (MC) pipettando il buffy coat (strato di cellule di barriera tra il gel e plasma) in un tubo da centrifuga sterile 15 ml. Portare il volume totale di 10 ml con soluzione salina tamponata con fosfato (PBS), invertito diverse volte e centrifugare a 300 xg per 15 min. </li><li> Risospendere le cellule in 10 ml di PBS sterile ed eseguire conta cellulare. Trasferire 1 a 2x10 6 cellule in una provetta da centrifuga sterile 15 ml conica e centrifugare a 300 xg per 10 min. </li><li> Risospendere le cellule in 2 ml di mezzo di espansione (EM) (QBSF-60 cellule Medium Stem contenente 50 ug / ml AscoAcid rbic, 50 ng / ml SCF, 10 ng / ml di IL-3, 2 U / ml EPO, 40 ng / ml IGF-1, 1 pM desametasone e 100 pg / ml primocin o 1% Pen / Strep) e trasferimento un pozzetto di una piastra da 12 pozzetti. Incubare le cellule a 37 ° C, 5% CO 2 incubatore. </li><li> Centrifugare le cellule rimanenti a 300 x g per 10 min e congelare ~ 2x10 6 cellule / flacone contenente FBS al 10% DMSO. </li><li> Per avviare il protocollo utilizzando PBMC congelato, scongelare 1 fiala di cellule in 10 ml di terreno QBSF e centrifugare a 300 xg per 10 min. Risospendere le cellule in 2 ml di EM e trasferire ad un pozzetto di una piastra da 12 pozzetti. Incubare le cellule a 37 ° C, 5% CO 2 incubatore. </li></ol> GIORNO 3 e 6 ° GIORNO <ol start="17"><li> Trasferire le cellule ad una sterile 15 ml tubo conico e lavare bene una volta con 1 ml di QBSF-60 Media delle cellule staminali per raccogliere le cellule aderenti. </li><li> Centrifugare le cellule a 300 xg per 10 min. </li><li> Risospendere le cellule in 2 ml di EM e trasferire ad un pozzetto di un 12-Pozzetti. Incubare le cellule a 37 ° C, 5% CO 2 incubatore. </li></ol> 2. Trasduzione di PBMC con STEMCCA Lentivirus GIORNO 9 <ol><li> Trasferire le cellule ad una sterile 15 ml tubo conico e lavare bene una volta con 1 ml di QBSF-60 Media delle cellule staminali per raccogliere le cellule aderenti. </li><li> Centrifugare le cellule a 300 xg per 10 min. </li><li> Risospendere le cellule in 1 ml di EM fresco contenente 5 mg / ml di polibrene e STEMCCA lentivirus (MOI = 1 a 10) e di trasferimento ad un pozzetto di una piastra da 12 pozzetti. (Il protocollo che descrive la produzione di particelle lentivirali sono disponibili all&#39;indirizzo <a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a> ) </li><li> Centrifugare la piastra a 2250 rpm a 25 ° C per 90 min. </li><li> Dopo la rotazione, aggiungere un ulteriore 1 ml di EM freschi contenente 5 mg / ml di polibrene per un totale di 2 ml di terreno e incubare la piastra a 37 ° C,5% di CO 2 incubatore. </li></ol> GIORNO 10 <ol start="16"><li> Trasferire le cellule ad una sterile 15 ml tubo conico e lavare bene una volta con 1 ml di QBSF-60 Media delle cellule staminali per raccogliere le cellule aderenti. </li><li> Centrifugare le cellule a 300 xg per 10 min. </li><li> Risospendere le cellule in 2 ml di EM e trasferire a 1 pozzetto di una piastra da 12 pozzetti. Incubare le cellule a 37 ° C, 5% CO 2 incubatore. </li></ol> 3. Placcatura cellule trasdotte sul MEF GIORNO 11 <ol><li> Cappotto dei pozzetti di una piastra da 6 pozzetti con 0,1% di gelatina e fibroblasti di topo piastra inattivati ​​embrionali (MEF) a 2x10 5 cellule / pozzetto in terreno MEF (IMDM contenente 10% FBS, 1% Aminoacidi non essenziali, 100 pM β- mercaptoetanolo, 2 mM L-glutammina, 100 pg / ml primocin o 1% Pen / Strep). Preparare 3 pozzetti per infezione. </li></ol> GIORNO 12 <ol start="2"><li> Trasferire le cellule ad una sterile 15 ml tubo conico<html> e lavare il bene una volta con 1 ml di QBSF-60 Media delle cellule staminali per raccogliere le cellule aderenti. </li><li> Risospendere le cellule in 3 ml di mezzo di MEF contenente 10 ng / ml bFGF, e l&#39;acido ascorbico e fattori di crescita alle stesse concentrazioni come utilizzato in EM media (50 ug / ml di acido ascorbico, 50 ng / ml SCF, 10 ng / ml IL -3, 2 U / ml EPO, 40 ng / ml IGF-1, 1 pM desametasone). </li><li> Piastra 1 ml di cellule per pozzetto di una piastra da 6 pozzetti contenente MEFs. Aggiungere 1,5 ml di MEF media con bFGF, acido ascorbico, e fattori di crescita per un totale di 2,5 ml di mezzo / e. </li><li> Centrifugare la piastra a 500 rpm a 25 ° C per 30 min. Incubare la piastra a 37 ° C, 5% CO 2 incubatore. </li></ol> GIORNO 14 <ol start="6"><li> Cellule di alimentazione a giorni alterni con 2,5 ml di mezzo di MEF contenenti 10 ng / ml bFGF e 50 ug / ml di acido ascorbico (non fattori di crescita). Aspirare ed eliminare le cellule mobili con ogni feed. </li><li> Aggiungi ulteriori MEF in base alle esigenze (di solito una volta alla settimana). </li></ol>p &quot;&gt; ~ GIORNO 20 <ol start="8"><li> Una volta che la comparsa di colonie, le cellule di alimentazione giornaliera con 2 ml di cellule staminali embrionali umane (hESC) medio (DMEM/F12 sostituzione contenente Knockout 20% siero, 1% amminoacidi non essenziali, 100 mM β-mercaptoetanolo, 2 mM L-Glutammina , 100 pg / ml primocin o 1% Pen / Strep e 10 ng / ml bFGF). </li></ol> 4. Raccolta ed espansione di cloni iPSC ~ DAY 30-40 <ol><li> Selezionare ogni colonia in singoli pozzetti di un 12-pozzetti pre-seminato con MEF inattivato contenente 1 ml / pozzetto di media hESC più 10 pM di Stemolecule Y27632 (ROCK inibitore). </li><li> Pasci le cellule quindi giornalmente con 1 ml di terreno hESC solo (senza inibitore ROCK). </li></ol> 5. Colorazione di immunofluorescenza per Marcatori pluripotenza <ol><li> La colorazione è stata eseguita utilizzando il kit &quot;Cell ES Kit Marker campione&quot; da Millipore e seguendo il protocollo del produttore. </li></ol> 6. Asportazione di Integnominale STEMCCA Vector <ol><li> Asportazione di riprogrammazione cassetta si ottiene utilizzando un Cre-IRES-Puro transfezione vettore che esprime seguendo e selezione puromicina breve come descritto 6. </li></ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Takahashi, K., Tanabe, K., Ohnuki, M., et al. <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., et al. <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 (2007).</li><li>Stadtfeld, M., Hochedlinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

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Abbiamo qui descrivere l&#39;uso del vettore lentivirale STEMCCA per generare iPSCs umani da cellule mononucleate isolate da pochi ml di sangue periferico raccolto di recente. Il protocollo può essere utilizzato anche per riprogrammare congelati (PBMC ottenute direttamente dal buffy coat), un particolare di implicazioni pratiche quando utilizza cellule donatrici acquisiti da un luogo distante. Prima dell&#39;induzione di riprogrammazione, PBMC isolate deve subire una fase critica di espansione che rende una popolazione...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> BD Vacutainer CPT Preparazione Tubo cellulare con citrato di sodio </td><td> BD Biosciences </td><td> 362760 </td><td></td></tr><tr><td> QBSF-60 cellule staminali Media </td><td> Di qualità biologica </td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td> Atlanta Biologicals </td><td> S10250 </td><td></td></tr><tr><td> Knockout sostituzione Siero </td><td> Invitrogen </td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> ant-pm-2 </td><td></td></tr><tr><td> Pen / Strep </td><td> Invitrogen </td><td> 15140 </td><td></td></tr><tr><td> L-Glutammina </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Aminoacidi non essenziali </td><td> Invitrogen </td><td> 11140 </td><td></td></tr><tr><td> β-mercaptoetanolo </td><td> MP Biomedicals </td><td> 190242 </td><td></td></tr><tr><td> Acido ascorbico </td><td> Sigma </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> R &amp; D Systems </td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R &amp; D Systems </td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R &amp; D Systems </td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R &amp; D Systems </td><td> 286-EP </td><td></td></tr><tr><td> Desametasone </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> Polibrene </td><td> Sigma </td><td> H-9268 </td><td></td></tr><tr><td> bFGF </td><td> R &amp; D Systems </td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES cellulare Marker Kit campione </td><td> Millipore </td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

Attraverso l&#39;espressione ectopica di quattro fattori di trascrizione, Oct4, Klf4, Sox2 e cMyc, cellule somatiche umane possono essere convertiti in uno stato pluripotente, generando le cosiddette cellule staminali pluripotenti indotte (iPSCs) 1-4. Paziente-specifici iPSCs mancano le preoccupazioni etiche che circondano le cellule staminali embrionali (ESC) e sarebbe possibile bypassare rigetto immunitario. Così, iPSCs hanno attirato molta attenzione per gli studi di modellazione delle malattie, la proiezione di composti farmacologici e terapie rigenerative 5. Abbiamo dimostrato la generazione di iPSCs umani transgene-liberi da pazienti con malattie polmonari diverse utilizzando un unico soggetto ad accisa policistronica cassetta lentivirale Stem Cell (STEMCCA), che codifica i fattori Yamanaka 6. Queste linee IPSC sono stati generati da fibroblasti della pelle, il tipo cellulare più utilizzato per la riprogrammazione. Normalmente, ottenendo fibroblasti richiede una biopsia cutanea seguita da espansione della cellas in coltura per alcuni passaggi. Importante, una serie di gruppi hanno riportato la riprogrammazione di cellule del sangue periferico in iPSCs 7-9. In uno studio, una versione inducibile Tet del vettore STEMCCA stato impiegato 9, che ha richiesto le cellule del sangue per essere simultaneamente infettati con un lentivirus costitutivamente attivo codificante l&#39;inverso transattivatore della tetraciclina. A differenza di fibroblasti, cellule del sangue periferico può essere smaltito attraverso procedure mini-invasive, riducendo notevolmente il disagio e la sofferenza del paziente. Un protocollo semplice ed efficace per la riprogrammazione delle cellule del sangue con un singolo vettore costitutivo soggetti ad accisa possono accelerare l&#39;applicazione della tecnologia iPSC rendendola accessibile ad una comunità di ricerca più ampio. Inoltre, la riprogrammazione delle cellule del sangue periferico consente la generazione di iPSCs da individui in cui biopsie cutanee dovrebbero essere evitati (ad esempio. Aberrante cicatrici) oa causa di condizioni pre-esistenti malattie Preventiaccesso ng a biopsie. Qui mostriamo un protocollo per la generazione di iPSCs umane da cellule mononucleate del sangue periferico (PBMC) servendosi di un unico soggetto ad accisa floxed-vettori lentivirali costitutivamente esprimere i 4 fattori. PBMC freschi o scongelati vengono espanse per 9 giorni come descritto 10,11 in mezzo contenente acido ascorbico, SCF, IGF-1, IL-3 e EPO prima di essere trasdotte con il lentivirus STEMCCA. Le cellule vengono poi piastrate su MEF e ESC-colonie, come possono essere visualizzati due settimane dopo l&#39;infezione. Infine, cloni selezionati sono espansi e testati per l&#39;espressione dei marcatori pluripotenza SSEA-4, Tra-1-60 e Tra-1-81. Questo protocollo è semplice, robusto e altamente coerente, fornendo un metodo affidabile per la generazione di iPSCs umani facilmente accessibile da 4 ml di sangue.

Dimostriamo un protocollo semplice ed efficace per la generazione di iPSCs da PBMC umane utilizzando un singolo vettore lentivirale. Figura 1A mostra una rappresentazione schematica del protocollo. Il sangue viene raccolto in una provetta BD Preparazione Vacutainer CPT cellulare con citrato di sodio e, dopo centrifugazione, le cellule mononucleate possono essere raccolte dall&#39;interfaccia tra il gel di poliestere e il plasma (buffy coat) (Figura 1B). Le PBMC isolate vengono espanse i...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Qui mostriamo un protocollo semplice ed efficace per la generazione di iPSCs umani da 3-4 ml di sangue periferico utilizzando un singolo vettore lentivirale riprogrammazione. La riprogrammazione delle cellule del sangue prontamente disponibili promette di accelerare l&#39;utilizzo della tecnologia iPSC rendendola accessibile ad una comunità di ricerca più ampio.

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, ...

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26.4K Views.  Boston University School of Medicine. Qui mostriamo un protocollo semplice ed efficace per la generazione di iPSCs umani da 3-4 ml di sangue periferico utilizzando un singolo vettore lentivirale riprogrammazione. La riprogrammazione delle cellule del sangue prontamente disponibili promette di accelerare l'utilizzo della tecnologia iPSC rendendola accessibile ad una comunità di ricerca più ampio.

Video: Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

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 ...

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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 ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Generazione rapida e efficiente di neuroni da cellule staminali umane pluripotenti in un formato piatto Multititre

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.