Questo lavoro è stato finanziato dalla Max Planck Society.

1. Preparazione di Matrigel piastre rivestite e Media <ol><li> Preparazione di Matrigel piastre rivestite Matrigel è stato trovato per essere un substrato preferito per il fissaggio di differenziare EBs e promuove anche la conseguenza efficiente di questi neuroni di 10. <ol><li> Scongelare 10 ml di una notte sul ghiaccio Matrigel. </li><li> Il giorno dopo, diluire il gelatinosa Matrigel con due volumi di ghiaccio-freddo DMEM/F-12 e preparare aliquote di 1 ml in prechilled provette da 15 ml coniche che possono essere conservati congelati a -20 ° C. </li><li> Decidere su un formato di piastra per la differenziazione neuronale. Per esempio, come punto di partenza, si consiglia di effettuare un primo giro di differenziazione in un 12-pozzetti. Pre-raffreddare quattro piastre da 12 pozzetti a 4 ° C. Sciogliere un&#39;aliquota di Matrigel congelate aggiungendo 9 ml di pre-raffreddate DMEM/F-12 seguiti pipettando su e giù fino a che tutto il prediluito Matrigel è stato scongelato e disciolto. </li><li> Poi, Trasferire il contenuto in un nuovo tubo da 50 ml contenente 15 ml di DMEM/F-12 su ghiaccio (diluizione finale Matrigel: 1:75). Quindi, si aggiungono 0,5 ml di Matrigel diluito ad ogni pozzetto di pre-raffreddamento 12-pozzetti. Avvolgere le piastre con Parafilm e lasciare riposare a notte RT. </li><li> Giorno successivo, trasferire piastre a 4 ° C. Piastre rivestite possono essere conservati per diverse settimane prima dell&#39;uso. </li></ol></li><li> Media </li></ol> EB medie formazione (~ 15 ml necessario effettuare differenziazione in una piastra a 96 pozzetti - vedi Tabella 1 e schema della figura 2): DMEM/F-12 con il 0,4% (PVA) 1x N2 1x B27 0,05% BSA 20 ng / ml FGF2 10 pM Y-27.632 1x PenStrepGln Differenziazione media (~ 30 ml necessario effettuare differenziazione in una piastra a 96 pozzetti, seguito dalla formazione neurone in uno Matrigel rivestita 12-pozzetti, vedi Tabella 1): DMEM/F-12 1x N2 <br/&gt; 1x B27 0,05% BSA 0,5 micron PD0325901 15 um SB431542 0,5 micron Dorsomorphin 1x PenStrepGln 2. EB Formazione forzata <ol><li> Grow hPSCs sotto i vostri preferiti alimentatore senza condizioni. Usiamo MEF condizionata medie su Matrigel rivestite piastre da 6 pozzetti 15. Tuttavia, i sistemi di coltura di altri come i media definiti fatti in casa o commerciale deve anche lavorare. Al momento di avviare un esperimento differenziazione, hPSCs dovrebbe essere sub-confluenti e attiva crescita. </li><li> Rimuovere meccanicamente spontaneamente colonie differenziate con una punta di pipetta sterile di plastica sotto un microscopio stereo. </li><li> Lavare le restanti colonie indifferenziate con PBS, e digerire di singole cellule utilizzando Accutase contenente 1X Y-27632. Pellet di cellule singole mediante centrifugazione a 200 xg per 2 minuti, risospendere in un piccolo volume di mezzo di formazione EB e determinare titolo cellulare mediante un hematocytometer. </li><li> Aggiungi una aliquota di celluleil restante formazione medio EB comportare un titolo tra 40.000 e 80.000 cellule per ml. </li><li> Usando una pipetta multicanale, trasferire 100 microlitri per pozzetto di una piastra di fondo-96V, con conseguente 4000 a 8000 cellule per pozzetto. Spin piastra da 96 pozzetti in un incernierato centrifuga piastra a 400 xg per 1 minuto per raccogliere le cellule al fondo dei pozzetti, e trasferimento incubatore per colture cellulari. EBs formerà durante la notte, come mostrato in Figura 1. </li></ol> 3. Neuroectoderma induzione <ol><li> Giorno successivo (giorno 0), raccogliere EBs dalla piastra 96V sotto un microscopio stereo e trasferimento in un piccolo volume in un piatto 3,5 centimetri batterica contenente 2 ml di terreno di differenziamento. Agitare delicatamente il piatto per alcuni secondi per lavare il EBS. </li><li> Aggiungere 100 pl di mezzo di differenziazione per pozzetto di un ultra-bassa attacco U-bottom piastra a 96 pozzetti. In uno stereomicroscopio, il trasferimento EBS in piccoli volumi dalla lavastoviglie in 96U-pozzetti (uno EB per noill). Inserire la piastra di 96U in incubatore per colture cellulare per 4 giorni per permettere la formazione neuroectoderma. </li></ol> 4. Neuron Formazione <ol><li> Il giorno 4, preparare un piatto di lavaggio come al punto 3.1 e inoltre sostituire DMEM/F-12 in un pre-riscaldato Matrigel rivestita 12-pozzetti da terreno di differenziamento (1 ml per pozzetto). </li><li> Placcatura di EBS <ol><li> Raccogliere EBs dalla piastra 96U, lavare questi semi come sopra, e con cura uno per uno nei pozzetti del Matrigel rivestite 12-pozzetti (circa 8 EBs per pozzetto): EB deve essere equamente distribuita all&#39;interno dei pozzetti per consentire di conseguenza indisturbato neuroni nel corso dei prossimi giorni (vedi immagine &quot;ore 5&quot; in Figura 2). </li><li> Prima del trasferimento del 12 e piastra posteriore in incubatrice, EBs consentono di collegare liberamente per circa 10 minuti a temperatura ambiente. Poi, trasferire accuratamente 12 pozzetti alla cultura incubatore cellulare. Neuroni crescerà fuori dal placcato EBs in modo radiale entro 4 giorni, come shown in figura 2 (il &quot;Giorno 8&quot; immagine a destra). </li></ol></li></ol> 5. Formazione Neuron come monostrati <ol><li> In alternativa alle fasi del punto 4), al giorno 4 della procedura, raccogliere EBs in un piatto 3,5 centimetri batterica contenente 2 ml di terreno di differenziamento, poi trasferire EBs in un piccolo volume di PBS contenente un piatto, e poi in un altro piatto contenente Accutase. </li><li> Lasciate digerire a cellule singole, centrifugare a 200 xg per 2 minuti, risospendere in un piccolo volume di terreno di differenziamento e determinare titolo cellulare mediante un hematocytometer. Regolare il titolo a 1-2X10 6 cellule per ml con terreno di differenziamento (senza Y-27632). </li><li> Cellule piastra out in pre-riscaldato Matrigel rivestite 12-pozzetti (1 ml per pozzetto), e trasferimento in coltura cellulare incubatore. Formazione neurone come monostrati è stata osservata tra i giorni 7-8 (Figura 3C). Va notato, tuttavia, che questa variante del monostratola procedura non è completamente ottimizzato per quanto riguarda la sopravvivenza delle cellule e substrato più adatto. </li></ol>

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Maggior parte dei protocolli di differenziazione che comportano la formazione di EB da hPSCs, compresa la nostra procedura precedentemente pubblicato con il 10, sono basati su manuale o enzima-assistita raccolta di hESC come aggregati, il che porta inevitabilmente a eterogeneità nei formati EB. Questo fatto porta a variabilità in efficienza differenziazione con alcune linee cellulari, rendendo così difficile analisi sistema, in particolare su scala velocità media. Inoltre, la dissezione manuale è alta in...

hPSCs, composto da embrioni derivati ​​da cellule staminali embrionali umane (hESC) e cellule staminali pluripotenti indotte (hiPSCs), sono pluripotenti significato che possono dare origine a diverse linee cellulari in vitro 1-2. Numerosi tipi di cellule differenziate sono stati derivati ​​da hESC ad oggi, sostenendo l&#39;idea che queste cellule presentano strumenti utili per la ricerca scientifica e basati su celle approcci di screening, e promettenti per terapie a base di cellule. Protocolli per la differenziazione neuronale hESC sono solitamente complesse e lunghe quanto sono spesso basati sulla prima sorte indurre complessiva neurale, seguito da isolamento manuale dei progenitori neurali, e la differenziazione terminale successiva in un determinato tipo di neurone di interesse 3-6. In alcuni riguardo, questa complessità non è sorprendente e inevitabile dato che un tale state-of-the-art protocolli di differenziazione diretti tendono a imitare graduale primi stadi dello sviluppo umano, which è di per sé estremamente complesso. D&#39;altra parte, può in parte anche riflettere la nostra mancanza di comprensione dei processi molecolari sottostanti, con conseguente protocolli di differenziazione che sono ancora lungi dall&#39;essere completamente ottimizzato. Per quanto riguarda la conversione di hPSCs indifferenziate in neuroectoderma, Pera et al. trovato che la soppressione di BMP / SMAD1/5/8 segnalazione aumenta l&#39;induzione neurale del hESC 7. Inoltre, l&#39;inattivazione di SMAD2 / 3 segnalazione ha dimostrato di promuovere la formazione neuroectoderma 8. Di conseguenza, l&#39;inattivazione combinata di queste due vie tende a generare una più efficace di induzione neurale del hPSCs 4,9. Più di recente, abbiamo dimostrato che un percorso di segnalazione terzo, FGF / ERK, funge da repressore forte delle prime fasi di impegno neuroectodermica in hESC. Al contrario, la repressione simultanea di tutti e tre questi percorsi portare a quasi omogenea conversione di hESC in neuroectoderma conin soli quattro giorni 10. Successivamente, la differenziazione terminale altamente efficiente in neuroni funzionali è stato osservato entro otto giorni. I neuroni ottenuti sono stati probabilmente i neuroni sensoriali del sistema nervoso periferico, che può in parte spiegare la loro derivazione rapida 10. Difetti nella manutenzione neurone sensoriale è considerata una causa di alcune patologie umane come disautonomia familiare 11. Per la modellazione di tali malattie basato su tecnologia hiPSC 12, per un&#39;ulteriore caratterizzazione funzionale, nonché per scopi applicati non richiedono alcun tipo particolare di neuroni, questa procedura differenziazione può presentare una base utile. Tuttavia, la strategia di differenziazione che avevamo originariamente impiegato coinvolto formazione di corpi embrionali (EB) da ciuffi di colonie hESC 10, e ciò ha determinato un bel grado di eterogeneità per quanto riguarda formati EB, e apparso anche di compromettere l&#39;efficienza formazione dei neuroni in alcune ceLinee ll. Inoltre, a causa della eterogeneità in dimensioni EB, la possibilità di verificare sistematicamente effetti dei fattori di crescita addizionali o piccole molecole durante e dopo la formazione dei neuroni sembravano essere alquanto confuso. In questo rapporto, abbiamo adattato la nostra procedura precedente per un rendimento medio-compatibile, l&#39;aggregazione forzata a base di tecnica per generare EBs in una dimensione altamente controllato modo, come mostrato anche in altri contesti 13. Successivamente, l&#39;EBS generato in forma di V pozzetti di piastre da 96 pozzetti sono state trasferite a forma di U bassissimo attaccamento piastre da 96 pozzetti, per avviare la formazione neuroectoderma in sospensione. Quattro giorni più tardi, l&#39;EBS potrebbe essere piastrate dando origine a neuroni terminalmente differenziate ad alta efficienza e omogeneità. In alternativa, giorno-4 EBs erano dissociate in singole cellule e placcato in quanto tale, che ha portato a bassa densità monostrati di neuroni umani. Risultati coerenti sono stati finora ottenuti con due indipendentemente delinee di hESC rived, HuES6 e NCl3. Come prerequisito, formazione EB successo era strettamente dipendente l&#39;uso di un polimero sintetico, alcool polivinilico (PVA), insieme con inibitore ROCK Y-27.632 noto per promuovere la sopravvivenza hESC 13-14. Come risultato, l&#39;omogeneità del EBs formata in 96-V-piastre è stata notevolmente migliorata rispetto a formazione di EBS come culture di massa basato su metodi di frazionamento casuali. Questo sistema fornisce quindi una piattaforma significativamente migliorato per la formazione neuroectoderma in coltura in sospensione, seguito dalla formazione neurone altamente controllato in condizioni aderenti.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Matrigel HC </td><td> Becton Dickinson </td><td> 354263 </td><td> Vedere il testo per ulteriori dettagli sulla gestione </td></tr><tr><td> DMEM/F-12 </td><td> Life Technologies </td><td> 21331-020 </td><td></td></tr><tr><td> Poli (vinil alcool) </td><td> Sigma </td><td> 363170 </td><td> Sciogliere al 0,4% in DMEM/F-12 utilizzando un bagno d&#39;acqua ad ultrasuoni / evitare il surriscaldamento / può essere conservato a 4 ° C per diverse settimane </td></tr><tr><td> N2 Supplemento </td><td> Life Technologies </td><td> 17502-048 </td><td> 100X, memorizzare come aliquote congelate </td></tr><tr><td> B27 Supplemento </td><td> Life Technologies </td><td> 12587-010 </td><td> 50X, negozio come aliquote congelate </td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> A1595 </td><td> 10% = 500X, negozio come aliquote congelate </td></tr><tr><td> L-Glutammina con penicillina / streptomicina </td><td> PAA </td><td> P11-013 </td><td> O equivalente </td></tr><tr><td> FGF2 </td><td> Peprotech </td><td> 100-18B </td><td> Sciogliere a 10 pg / ml in 0,1% di BSA / PBS = 1000X, negozio come aliquote congelate </td></tr><tr><td> ROCK inibitore Y-27.632 </td><td> abcamBiochemicals </td><td> Asc-129 </td><td> Sciogliere a 10 mM in DMSO = 1000X, negozio come aliquote congelate </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> O equivalente </td></tr><tr><td> Accutase </td><td> PAA </td><td> L11-007 </td><td></td></tr><tr><td> Da 96 pozzetti con fondo a V piastre </td><td> Nunc </td><td> 277143 </td><td></td></tr><tr><td> PD0325901 </td><td> Axon medchem </td><td> Axon 1408 </td><td> Sciogliere a 0.5 mM in DMSO = 1000X, negozio come aliquote congelate </td></tr><tr><td> SB431542 </td><td> abcamBiochemicals </td><td> Asc-163 </td><td> sciogliere a 15 mM in DMSO = 1000X, negozio come aliquote congelate </td></tr><tr><td> Dorsomorphin </td><td> Santa Cruz </td><td> sc-200689 </td><td> sciogliere a 0.5 mM in DMSO = 1000X, negozio come aliquote congelate </td></tr><tr><td> 96 pozzetti U-basso ultra-low di fissaggio piastre </td><td> Corning </td><td> 7007 </td><td></td></tr><tr><td> beta-III anticorpo Tubulin (mouse) </td><td> Sigma </td><td> T8660 </td><td> utilizzare a 1:1000 </td></tr><tr><td> beta-III tubulina (coniglio) </td><td> Covance </td><td> PRB-435P </td><td> utilizzare a 1:2000 </td></tr><tr><td> BRN3A (POU4F1) anticorpo </td><td> Santa Cruz </td><td> sc-8429 </td><td> utilizzare a 1:500 </td></tr><tr><td></td><td></td><td></td><td> Tabella 1. &lt;/ Strong&gt; reagenti e attrezzature specifiche. </td></tr></tbody></table>

rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format

Protocolli esistenti per la generazione di neuroni da cellule staminali pluripotenti (hPSCs) sono spesso noioso in quanto sono procedure multistep coinvolgono l&#39;isolamento e l&#39;espansione di cellule precursori neurali, prima differenziazione terminale. In confronto a questi approcci in termini di tempo, abbiamo recentemente scoperto che l&#39;inibizione combinata di tre vie di segnalazione, TGFβ/SMAD2, BMP/SMAD1 e FGF / ERK, promuove la rapida induzione neuroectoderma da hPSCs, seguita da immediata differenziazione in neuroni funzionali. Qui, abbiamo adattato la nostra procedura per un nuovo formato di piastra multititre, per migliorare ulteriormente la sua riproducibilità e di renderlo compatibile con metà del throughput delle applicazioni. Esso si articola in quattro giorni di formazione neuroectoderma in sfere galleggianti (corpi embrionali), seguito da altri quattro giorni di differenziamento in neuroni in condizioni aderenti. La maggior parte delle cellule ottenute con questo protocollo sembrano essere i neuroni sensoriali bipolari. Inoltre,la procedura è altamente efficiente, non richiede particolari competenze esperte, e si basa su un mezzo chimicamente definito semplice con piccole molecole costo-efficienti. A causa di queste caratteristiche, la procedura può servire come piattaforma utile per un&#39;ulteriore indagine funzionale così come per la cella di screening avvicina richiedendo umani neuroni sensoriali o neuroni di qualsiasi tipo.

Nella nostra precedente relazione, in linea con molti altri, EBs da hPSCs potrebbe essere generato semplicemente replating aggregati al momento scissione di routine di hPSCs in basso attaccamento piatti 10. Questo di solito si traduce in una distribuzione gamma di formati EB risultanti (Figura 1C, in alto). Un altro problema derivante da questo è che EBs mantenute in sospensione tendono ad aggregarsi tra loro, portando ad una eterogeneità ancora maggiore di dimensioni EB. Per aggirare quest...

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

Protocolli per la differenziazione neuronale di cellule staminali pluripotenti umane (hPSCs) sono spesso molto tempo e richiedono notevoli capacità di colture cellulari. Qui, abbiamo adattato una piccola molecola procedura basata differenziazione di un formato di piastra multititre, permettendo la generazione semplice, rapido, efficace e di neuroni umani in modo controllato.

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 ...

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43.5K Views.  Max Planck Institute for Molecular Biomedicine. Protocolli per la differenziazione neuronale di cellule staminali pluripotenti umane (hPSCs) sono spesso molto tempo e richiedono notevoli capacità di colture cellulari. Qui, abbiamo adattato una piccola molecola procedura basata differenziazione di un formato di piastra multititre, permettendo la generazione semplice, rapido, efficace e di neuroni umani in modo controllato.

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

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 ...

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Biologia

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rapido ed efficiente Zebrafish genotipizzazione Uso PCR con ad alta risoluzione Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

Sporulazione efficiente di<em&gt; Saccharomyces cerevisiae</em&gt; In un pozzetti formato 96

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

Generazione efficiente e coerente di epitelio/coroide pigmentato retinico Flatmounts da occhi umani per analisi istologiche

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...