Questo lavoro è stato sostenuto dal New York State Department of Health (C026878). MS è stato a New York Stem Cell Foundation-Druckenmiller Fellow. Sostenuto in parte da assegno di ricerca n ° 5-FY11-571 dal March of Dimes Foundation.

1. Preparazione di cellule feeder Si noti che tutti i reagenti descritti di seguito devono essere preparati in modo sterile (vedi tavole 1 e 2). Questo protocollo utilizza la linea JK1 cella (Cell Biolabs, Inc., catalogo # CBA-315) come alimentatori che è un derivato trasformata di topo adulto testicolari cellule somatiche ed è stato descritto altrove 3. Si noti inoltre che tutte le procedure di polizia devono essere eseguite in conformità con le linee guida istituzionali e dei regolamenti. <ol><li> JK1 cellule possono essere mantenute in coltura e usato con successo come alimentatori fino al passaggio 39. Cultura JK1 cellule in un piatto di coltura cellulare 100 mm (BD Falcon, catalogo # 353.003) con 10 ml di mezzo alimentatore sterilizzata a filtro crescita (DMEM supplementato con 10% di siero fetale bovino [FBS], 2 mM L-glutammina e antibiotici). Quando la cultura raggiunge il 95% di confluenza, dividere le celle 1:06 -1:10 rapporto. </li><li> Estrarre il supporto dal confluente JK1 mono alimentatore cellastrato. </li><li> Lavare strato di cellule una volta con PBS senza calcio e magnesio. </li><li> Aggiungi preriscaldato tripsina / EDTA, 1x (0,05% trypsin/0.53 mM EDTA; Corning Cellgro, catalogo # 25-051-CI), e incubare a 37 ° C per 5-10 minuti. </li><li> Inattivare la tripsina con un volume uguale di mezzo di crescita alimentatore. Raccogliere le cellule e centrifugare a 300 xg per 5 min. Risospendere le cellule in terreno di crescita alimentatore. </li><li> Pre-coat multi-pozzetti piastre cella aggiungendo abbastanza 0,4% soluzione di gelatina per coprire il bene e togliere dopo 5 min a temperatura ambiente. </li><li> Piatto ~ 250 cellule per millimetro 2 in gelatina rivestita con piastra di coltura cellulare (ad esempio, in formato a 48 pozzetti o 6-bene). Incubare coltura a 37 ° C per 16 a 24 ore. </li><li> Togliere terreno di coltura da pozzi. ATTENZIONE: Per inattivare crescita cellulare, aggiungere una soluzione fresca di mitomicina-C a 10 ug / ml diluito in DMEM a ciascun pozzetto (200 pl / pozzetto di una piastra da 48 pozzetti). Mitomicina-C è tossico. Maneggiate e smaltite con cura. Incubare a37 ° C per 4 h. </li><li> Rimuovere mitomicina-C soluzione da cellule. Lavare 3 volte con DMEM prima placcatura cellule staminali. </li><li> Rimuovere DMEM dall&#39;alimentatore cellule pozzetti e aggiungere abbastanza cellule staminali media (100 pl / pozzetto di una piastra da 48 pozzetti, vedi Tabella 3) per prevenire l&#39;essiccamento delle mangiatoie durante passaging. Aggiungi CSS lo stesso giorno, come descritto di seguito nelle sezioni 3.1 o 3.4, utilizzando le dimensioni indicate di pozzetti contenenti alimentatori. </li></ol> 2. La dissociazione del tessuto mouse Testicolo ottenere cellule germinali dei donatori <ol><li> Harvest testicoli di topo adulto in modo sterile e memorizzare temporaneamente i testicoli in una capsula di Petri coperto (senza liquido aggiunto). Il piatto deve posto immediatamente in ghiaccio per evitare essiccazione e mantenere la vitalità cellulare. </li><li> Utilizzando una pinza sottile sterili e una forbice fine in una cappa di coltura cellulare, fare una incisione trasversale nella tunica albuginea completamente senza sezionare il testicolo (cioè tagliato ~ ½ - ¾ distanza through) e usare il forcipe per spremere tubuli seminiferi in un angolo della piastra, tunica degli scarti e dei tessuti allegato. Tenere piastra su ghiaccio in tutto per tenere il tessuto fresco, mantenendo così la sua integrità e prevenire l&#39;evaporazione. </li><li> Rapidamente tritare tubuli con le forbici a molla sottili almeno 3 minuti al testicolo, mantenendo piastra su ghiaccio. </li><li> Raccogliere i frammenti di tubuli in un tubo da 50 ml e lavare in ~ 40 ml di siero refrigerati PBS / 1% di albumina bovina. </li><li> Centrifugare a 60 xg per 10 min per separare frammenti tubulari da spermatozoi e detriti. Gettare il surnatante. </li><li> In una provetta conica da 15 ml, risospendere il pellet in 3 ml per testis di pre-riscaldato dissociazione buffer (DMEM, 0,05% tripsina, collagenasi tipo I 0,03%, 80% U / ml di DNasi I e 0,5 albumina di siero bovino; Vedi Tabella 2 per i dettagli più specifici di dissociazione buffer). </li><li> Posizionare il tubo conico orizzontalmente in un rack in un insieme agitatore a 150 rpm a 37 ° C per 15 minuti per massimizzare unagitation. </li><li> Centrifugare a 60 xg per 10 min per separare singole cellule di pezzi non dissociato. </li><li> Salvare i pezzi di pellet contenenti fino alla fase successiva. Raccogliere il supernatante dal passo 2,8 e aggiungere 3 ml DMEM/10% di siero fetale bovino per neutralizzare buffer di dissociazione. Porre la provetta in ghiaccio temporaneamente. </li><li> Ripetere il passaggio 2,6-2,7 con il pellet salvato dal punto 2.9 a ri-digerire pezzi rimanenti dopo la fase 2.8. </li><li> Aggiungere un volume uguale di DMEM/10% FBS per neutralizzare tampone di dissociazione. </li><li> Sospensione ricombinare dal punto 2,11 con sospensione cellulare salvato dal punto 2.9. Centrifugare per 5 minuti a 300 x g. </li><li> Risospendere il pellet in media di cellule staminali in un volume di 16 ml per ogni coppia di testicoli (vedi Tabella 3). </li></ol> 3. Placcatura di sospensione cellulare testicolare <ol><li> Piastra 2 ml di sospensione cellulare per pozzetto in un 6-pure cellule feeder piastra contenente mitoticamente-inattivato con mitomicina-C e posto in un incubatore per colture cellulari a 37 &amp;deg; C in 5% CO 2. </li><li> Dopo 48 ore, mezzo aspirato, aggiungere 2 ml di terreno fresco e tornare alla incubatrice. </li><li> Dopo 48 ore, aggiungere 0,5 ml di terreno fresco di cellule staminali in ciascun pozzetto e tornare alla incubatrice. </li><li> Dopo 48 ore, successivamente nutrire le cellule tre volte alla settimana, successivamente segue come di grandi dimensioni, fino a quando le colonie discrete (&gt; 50 cellule) appaiono (entro 7 a 21 giorni). Per prima e la seconda alimentazione di ogni settimana (ad esempio, Lunedi e Mercoledì) rimuovere circa il 50% del mezzo e aggiungere di nuovo il 50% in volume di terreno fresco. Per l&#39;alimentazione terzo (ad es Venerdì), aspirare tutte di medie e sostituire con 2 ml di terreno fresco. Questo approccio si basa sul razionale che completi evitare cambiamenti medi minimizza le fluttuazioni nelle concentrazioni di segnali paracrini che vengono secrete nel mezzo di coltura 9. </li></ol> 4. Picking Colony per primo passaggio <ol><li> Estrarre il supporto dalle colonie pozzi contenenti da diversi passaggi e aggiungere fresco medio di cellule staminali. </li><li> Identificare le colonie con un obiettivo 10x. Con il microscopio leggermente de-focalizzato, colonie SSC a bordo del pozzo apparirà come macchie luminose omogenee, comprendenti cellule 11-12 micron molti diametro, in cui è difficile o impossibile distinguere i confini delle singole celle, poiché le cellule appaiono fusi insieme (Figura 1A). Non SSC colonie possono apparire più scuro, più granulare, o meno omogeneo, con bordi distinguibili (Figura 1A, nel riquadro). </li><li> Utilizzando una punta di 200 microlitri pipetta, con il set pipetteman a 50 microlitri, togli prima la media dal pozzo ed espellere a bagnare la punta. Poi, delicatamente spostare la colonia con la punta prima di ritirare rapidamente 50 microlitri di terreno di spostare la colonia di aspirazione nella punta (Figura 1B). </li><li> Espellere il supporto contenente la colonia in un pozzetto di una piastra a 48 pozzetti contenenti cellule feeder inattivate con 100 pl di mezzo di cellule staminali (Figura 1C). </li><li> Ripetere il passaggio4.3 e aggiungere fino a 8 colonie per il bene stesso, senza superare 500 microlitri per pozzetto. </li><li> Ripetere i passaggi 4,3-4,5 per preparare pozzi della piastra da 48 pozzetti con passaggio uno colonie SSC. </li><li> Wells sarà pronto a dividere in 7-14 giorni o dopo grandi gruppi di fino a 500 cellule sono emerse (Figura 1D). </li></ol> 5. Successiva espansione e passaging di SSC per triturazione <ol><li> Cellule al passaggio uno dovrebbe essere subcoltura su alimentatori fresche dopo fino a 14 giorni a una rapporto di divisione di 1:2 per i primi 3 passaggi e poi 1:04-01:06 (ogni 7 giorni) successivamente come segue. Delicatamente triturare colonie fuori un semi-confluente pozzetto (es. ~ 300.000 SSC / pozzetto della piastra da 6 pozzetti) con una punta di pipetta 1 ml lavando media tra le celle. Le colonie possono essere visti distacco. Forzare troppo con il flusso di liquido causerà cellule feeder indesiderate a venire fuori troppo. </li><li> Raccogliere cellule triturato in una provetta conica e centrifugare a 300 xg per5 min. Nota: Un passo tripsinizzazione può essere tranquillamente aggiunto qui, a discrezione del ricercatore. </li><li> Aspirare il surnatante e risospendere in mezzo fresco di cellule staminali da fondo pipettando su e giù per distruggere le colonie. </li><li> SSC piastra su cellule di alimentazione preparati al momento inattivati ​​con mitomicina-C, come descritto nella sezione 1. </li><li> Alimentare cellule tre volte alla settimana come segue. Per l&#39;alimentazione prima e la seconda (ad esempio, Lunedi e Mercoledì) aggiungere 50% in volume di terreno fresco. Per l&#39;alimentazione terzo (ad es Venerdì), aspirare tutte di medie e sostituire con 2 ml di terreno fresco. Le piastre saranno confluenti in 7-10 giorni. Le colture devono essere costituiti da cellule germinali&gt; 98% (vale a dire, &lt;1% di cellule somatiche contaminanti) dopo passaggi 5-7, basato su immunocolorazione (ad esempio, utilizzando il murino della cellula germinale marcatore GCNA) per distinguere le cellule germinali a partire da cellule somatiche 10. Nota: Questi metodi sono stati sviluppati in conformità alle norme e ai requisiti stabiliti dall&#39;IstituzioneCura degli animali e al comitato di uso al Weill Cornell Medical College. </li></ol>

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MS riceve canoni da Weill Cornell Medical College di attraverso una licenza to Cell Biolabs, Inc. per la distribuzione JK1 cellule.

Questo metodo per derivare SSC adulti con adulti testicolo derivate da cellule di alimentazione è robusto ed è riuscita quando comuni background genetico (ad esempio FVB, C57Bl6 e misti 129SV/C57Bl/6) e diversi ceppi mutanti sono stati impiegati 2,3,7,14. Infatti, il microambiente creato dal sistema di coltura è sufficiente a superare alcune delle barriere genetiche per mantenere SSCs adulti in vivo (ad esempio nel caso di PLZF - / - animali) 7. Mentre impieghiamo JK...

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> DMEM </td><td> Corning </td><td> 10-013 </td><td> Diluente per la dissociazione del buffer </td></tr><tr><td> Tripsina / EDTA </td><td> Mediatech </td><td> 25-051-CI </td><td></td></tr><tr><td> Staminali base medio cellulare (StemPro-34) </td><td> Life Technologies </td><td> 10639-011 </td><td> Richiede l&#39;integrazione come da Shinohara et al. (2003) * </td></tr><tr><td> Staminali integratori medie cellulari </td><td> vario </td><td> vedi Tabella 3 </td><td> Richiede l&#39;integrazione come da Shinohara et al. (2003) * </td></tr><tr><td> JK1 cellule </td><td> Cella Biolabs, Inc. </td><td> CBA-315 </td><td> Può sostituire con adulti cellule testicolari stromali come da Seandel et al. (2007) </td></tr><tr><td> mitomicina-C (ATTENZIONE) </td><td> Sigma-Aldrich </td><td> M4287 </td><td> Tossico, Maneggiare con cura. </td></tr><tr><td> Gelatina </td><td> Sigma-Aldrich </td><td> G1890 </td><td> 0,4% soluzione in acqua </td></tr><tr><td> EVOS xl digitale microscopio invertito </td><td> Gruppo Microscopia Avanzata </td><td> - </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 1. Reagenti e attrezzature specifiche. * Vedi Tabella 3 </td></tr><tr><td> DMEM </td><td> Corning </td><td> 10-013 </td><td> Diluente per la dissociazione del buffer </td></tr><tr><td> tripsina (1:250) </td><td> Life Technologies </td><td> 27250-018 </td><td> Dissociazione buffer: finale 0,05% peso / vol </td></tr><tr><td> collagenasi di tipo I, 235 U / ml </td><td> Worthington </td><td> CLS1 235 </td><td> Dissociazione buffer: finale 0,03% peso / volume </td></tr><tr><td> DNAsi I </td><td> SIGMa-Aldrich </td><td> DN25 </td><td> Dissociazione buffer: Finale 80 U / ml </td></tr><tr><td> albumina di siero bovino </td><td> ICP Bio </td><td> ABRE-100g </td><td> Dissociazione buffer: Finale 0,5% peso / volume </td></tr><tr><td></td><td></td><td></td><td> Tabella 2. Dissociazione tampone </td></tr><tr><td> StemPro-34 SFM </td><td> Life Technologies </td><td> 10639-011 </td><td></td></tr><tr><td> StemPro-34 supplemento nutriente </td><td> Life Technologies </td><td> 10639-011 </td><td></td></tr><tr><td> Supplementi aggiuntivi ** </td><td></td><td></td><td></td></tr><tr><td> Aminoacidi non essenziali </td><td> Sigma-Aldrich </td><td> M7145 </td><td> 1X </td></tr><tr><td> MEM Vitamina soluzione </td><td> Life Technologies </td><td> 11120-052 </td><td> 1X </td></tr><tr><td> L-glutammina </td><td> Mediatech </td><td> 25-005 </td><td> 2 mM </td></tr><tr><td> albumina di siero bovino </td><td> ICP Bio </td><td> ABRE </td><td> 0,50% </td></tr><tr><td> Antibiotico-antimicotica Solution </td><td> Mediatech </td><td> 30-004-CI </td><td> 1X </td></tr><tr><td> D (+) glucosio </td><td> Sigma-Aldrich </td><td> G8769 </td><td> 6 mg / ml </td></tr><tr><td> β-estradiolo </td><td> Sigma-Aldrich </td><td> E2758 </td><td> 30 ng / ml </td></tr><tr><td> progesterone </td><td> Calbiochem </td><td> 5341 </td><td> 60 ng / ml </td></tr><tr><td> siero fetale bovino </td><td> variabile </td><td> n / a </td><td> 1% </td></tr><tr><td> bovina olo-transferrina </td><td> Sigma-Aldrich </td><td> T1283 </td><td> 100 pg / ml </td></tr><tr><td> insulina </td><td> Gemini Bio-Prodotti </td><td> 700-112P </td><td> 25 mg / ml </td></tr><tr><td> umano GDNF </td><td> Vita Techndologie </td><td> PHC7041 </td><td> 10 ng / ml </td></tr><tr><td> umana bFGF </td><td> Life Technologies </td><td> PHG0023 </td><td> 10 ng / ml </td></tr><tr><td> del mouse FEG </td><td> Life Technologies </td><td> PHG0313 </td><td> 20 ng / ml </td></tr><tr><td> putrescina </td><td> La ricerca Organics </td><td> 0778P </td><td> 60 pM </td></tr><tr><td> Selenite di sodio </td><td> Sigma-Aldrich </td><td> S5261 </td><td> 30 nM </td></tr><tr><td> acido piruvico </td><td> Alfa Aesar </td><td> A13875 </td><td> 30 ug / ml </td></tr><tr><td> DL-lattico </td><td> JT Baker </td><td> 0196-04 </td><td> 1 pg / ml </td></tr><tr><td> β-mercaptoetanolo </td><td> Life Technologies </td><td> 21985-023 </td><td> 50 pM </td></tr><tr><td> acido ascorbico </td><td> Sigma-Aldrich </td><td> A4544 </td><td> 100 pM </td></tr><tr> &lt;td&gt; D-biotina </td><td> Sigma-Aldrich </td><td> B4639 </td><td> 10 ug / ml </td></tr><tr><td></td><td></td><td></td><td> Tabella 3. Staminali media cella </td></tr><tr><td></td><td></td><td></td><td> * Nota: Aggiungere i supplementi di seguito prima di utilizzare il terreno. Filtro sterilizzare e mantenere a 4 ° C. Il medium è stabile per almeno 2 settimane. </td></tr><tr><td></td><td></td><td></td><td> ** Abbiamo impiegato diversi produttori, formulazioni e / o numeri di lotto di reagenti, senza apparenti effetti deleteri. In generale, i reagenti di grado coltura cellulare dovrebbe essere impiegato. </td></tr></tbody></table>

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

Staminali spermatogoni e cellule progenitrici (SSC) del testicolo rappresentano un classico esempio di cellule staminali di mammifero e preservare la fertilità per quasi tutta la vita dell&#39;animale. Mentre i precisi meccanismi che governano self-renewal e la differenziazione in vivo sono difficili da studio, vari sistemi sono stati sviluppati in precedenza per propagare SSC murini in vitro utilizzando una combinazione di terreni di coltura e le cellule specializzate alimentazione 1-3. La maggior parte in incursioni in vitro sulla biologia della SSC hanno linee cellulari derivate da neonati, probabilmente a causa della difficoltà di ottenere linee di cellule adulte 4. Tuttavia, il testicolo continua a maturare fino a ~ 5 settimane di età in ceppi maggior parte dei mouse. Nel primo periodo post-natale, si verificano cambiamenti drammatici nell&#39;architettura dei testicoli e nella biologia delle cellule somatiche e sia spermatogenesi, incluse alterazioni nei livelli di espressione di cellule staminali numerosi correlatageni. Pertanto, neonatale-derivati ​​linee SSC potrebbero non ricapitolare la biologia della SSC adulti che persistono dopo che il testicolo adulto ha raggiunto uno stato stazionario. Diversi fattori hanno ostacolato la produzione di linee per adulti SSC storicamente. In primo luogo, la proporzione di cellule staminali funzionali può diminuire durante l&#39;età adulta, a causa di fattori intrinseci o estrinseci 5,6. Inoltre, come con altre cellule staminali adulte, è stato difficile per arricchire SSCs sufficientemente da cellule adulte totale testicolari senza utilizzare una combinazione di immunoselezione o altre strategie di smistamento 7. Strategie comunemente impiegati comprendono l&#39;uso di topi criptorchidi come fonte di cellule del donatore a causa di un alto rapporto di cellule staminali per altri tipi di cellule 8. Basata sull&#39;ipotesi che la rimozione di cellule somatiche dalla coltura iniziale interrompe interazioni con la nicchia delle cellule staminali che sono essenziali per la sopravvivenza SSC, abbiamo precedentemente sviluppato metodi per derivare l adultoines che non richiedono immunoselezione o donatori criptorchidi ma piuttosto impiegano arricchimento di serie SSC nella cultura, di seguito denominata SESC 2,3. Il metodo descritto di seguito comporta una semplice procedura per derivare adulti linee SSC dissociandosi adulto donatore tubuli seminiferi, seguito da placcatura su linee di cellule comprendenti una linea di cellule stromali testicolare (JK1) 3. Attraverso seriale passaging, fortemente aderente, contaminando le cellule germinali non sono esaurite dalla coltura con arricchimento concomitante di SSC. Culture prodotti in questo modo contengono una miscela di spermatogoni a diversi stadi di differenziazione, che contengono SSCS, basato su lungo termine capacità di rinnovamento del sé. Il punto cruciale del metodo SESC è che consente CSS per rendere la difficile transizione da self-renewal in vivo a lungo termine di auto-rinnovamento in vitro in un microambiente radicalmente diverso, produce a lungo termine linee SSC, libero di contaminarecellule somatiche, e consentendo in tal modo la successiva manipolazione sperimentale di SSC.

La comparsa di passaggio per lo zero di tipo selvatico, adulti colonie SSC dopo 7 giorni è mostrato in Figura 1. Tridimensionali colonie sono costituiti da uno strato di cellule piatte attaccate agli alimentatori o della matrice extracellulare underling depositati dagli alimentatori con strati multipli di SSCs crescono sopra. Mentre SSCs sani sono luminose e rifrangenti uniformemente 11-12 micron di diametro, i bordi delle celle sono difficili da distinguere e la dimensione delle colonie possono essere...

Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Un metodo semplice per ottenere e mantenere spermatogoni staminali e linee di cellule progenitrici da topi adulti è presentato qui. Il metodo utilizza cellule di alimentazione provenienti dal compartimento delle cellule somatiche del testicolo di topo adulto. Questa tecnica è applicabile ai ceppi di topi comuni, tra cui transgenici, knock-out e knock-nei topi.

Arricchimento di serie staminali spermatogoni e cellule progenitrici (SSC) in Cultura per la derivazione di lungo termine Lines topo adulto SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

serial-enrichment-spermatogonial-stem-progenitor-cells-sscs-culture

Research

JoVE Journal

Biology

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

13.7K Views.  Weill Cornell Medical College. Un metodo semplice per ottenere e mantenere spermatogoni staminali e linee di cellule progenitrici da topi adulti è presentato qui. Il metodo utilizza cellule di alimentazione provenienti dal compartimento delle cellule somatiche del testicolo di topo adulto. Questa tecnica è applicabile ai ceppi di topi comuni, tra cui transgenici, knock-out e knock-nei topi.

Video: Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Ricerca

Biologia

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasti derivazione dal midollo osseo di topo

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...