Il lavoro del laboratorio autori è stata sostenuta da USDA / CSREES / NRICGP (2007-35203-18213); NIH / NCRR (2 RR17359 R01-06), NIH / NIEHS (1 R21 ES014856-01A2) e Alberta Innovates - Health Solutions.

Germe di trapianto di cellule A. PART nei topi 1. Preparazione di topi riceventi <ol><li> I destinatari devono essere immunologicamente tolleranti (sia geneticamente identiche al donatore o con deficit immunitario) alle cellule del testicolo il donatore. </li><li> I destinatari devono essere naturalmente privo di spermatogenesi (ad esempio w / w v topi) o impoverito di cellule germinali endogene. Deplezione delle cellule di germe può essere raggiunto da radiazioni o da farmaci chemioterapici come il Busulfan. In questo protocollo, si descrive il metodo di preparazione di topi riceventi con Busulfan. </li><li> Trattare i destinatari a 4-6 settimane di età mediante iniezione ip di busulfan. La dose ottimale di busulfan è sforzo dipendente. In ceppi beneficiarie comunemente utilizzati, una dose di 40-50 mg / kg è sufficiente deplezione delle cellule germinali endogene (ad esempio 44 mg / kg per topi nudi, 50 mg / kg per B6/129 destinatari F1). </li><li> Sciogliere Busulfan polvere in DMSO e quindi aggiungere un uguale volume di sterile t acqua distillataØ fare una concentrazione finale di 4 mg / ml. Mantenere soluzione calda a 35-40 ° C prima dell&#39;uso per evitare la precipitazione di busulfan. Eliminare la soluzione se si osserva la precipitazione. </li><li> Dopo il trattamento Busulfan, lasciare almeno un mese prima di utilizzare i destinatari. I destinatari possono essere utilizzati tra 1 mese e 3 mesi post-trattamento Busulfan. </li></ol> 2. Preparazione di pipette microiniezione <ol><li> Scegliere pipette in vetro borosilicato (tubi capillari) con un diametro 1,0 millimetri esterno, uno diametro 0,75 mm interno, e una lunghezza di 3 pollici. </li><li> Siliconize pipette di vetro con Sigmacote, lavare pipette con metanolo e asciugare. </li><li> Tirare le pipette con un estrattore pipetta. Diverse macchine estrattori pipette hanno impostazioni diverse, di conseguenza, verificare alcune impostazioni. Generalmente, un&#39;impostazione simile a quella utilizzata per rendere pipette enucleazione può funzionare. </li><li> Rompere i punte di pipetta sotto microscopio da dissezione prima di utilizzare per raggiungere un diametro di circa 50 umsulla punta. </li></ol> 3. Preparazione di cellule del donatore per il trapianto <ol><li> Choise di ceppo donatore è dipende dalla questione sperimentale studiato. Se la quantificazione della spermatogenesi viene utilizzato come endpoint, l&#39;uso di donatori con un marker genetico facilmente identificabile come ad esempio Lac-Z (ad esempio B6.129S7-Gt (ROSA) 26Sor / J dal Jackson Laboratory) o GFP (ad es C57BL/6-Tg ( CAG-EGFP) 1Osb / J dal Jackson Laboratory). </li><li> Preparare 7 ml di collagenasi in DMEM a 1 ml mg / ml, 4 ml di tripsina-EDTA (0,25% trypsin più 1 mM EDTA), e 2 di DNase in DMEM a 7 mg / ml. Tale importo è per digerire 2 testicoli di topo donatori. </li><li> Raccogliere testicoli in modo asettico e togliere la tunica albuginea. Diffusione tubuli seminiferi delicatamente con una pinza sottile per facilitare la digestione enzimatica. </li><li> Tubuli trasferimento in collagenasi, incubare a 37 ° C per 5-10 minuti ed agitare spesso. </li><li> Lavare tubuli due volte da spinning (200-300 g per 3 min) e ri-sospensione in PBS w / o Ca 2 + + </sup&gt;. </li><li> Risospendere tubuli in tripsina-EDTA e agitare fino a farli diventare appiccicoso e poco nuvoloso. Monitorare la digestione di tubuli a 37 ° C come dovrebbe accadere entro 1-2 min. </li><li> Aggiungi DNasi e agitare bene. Incubare per 1-2 min. </li><li> Aggiungere 3 ml di FBS fermare l&#39;azione di enzimi. </li><li> Sospensione cellulare filtro con una maglia in nylon con 40-70 micron dimensioni dei pori per rimuovere cellule / tessuti pezzi. </li><li> Raccogliere le cellule a 600 g per 5 min e risospendere cellule in una piccola quantità di DMEM (&lt;100 pl). </li><li> Contare le cellule e regolare il volume ad una concentrazione finale desiderata (in genere 100 x 10 6). Mantieni sospensione cellulare sul ghiaccio prima dell&#39;uso. </li><li> Ogni Busulfan-trattati con testicolo mouse sarà riempito con solo il 10-15 microlitri di sospensione cellulare. A causa della potenziale spreco e perdita, mirano ad avere 30-50 pl per testicolo. </li></ol> 4. Transplantation procedura (Figura 1) <ol><li> Anestetizzare seguenti destinatari protocolli approvati. </li><li> Luogo in dorsaledecubito e chirurgicamente preparare l&#39;area addominale. Fai una ~ 1 centimetro incisione addominale mediana per esporre la parete addominale. Sollevare parete addominale utilizzando una pinza di piccole dimensioni in corrispondenza del punto della linea bianca per evitare di ferire accidentalmente organi addominali, e procedere per fare un 0,5 cm ~ incisione sulla linea mediana della parete addominale per esporre la cavità peritoneale. </li><li> Utilizzare una pinza dell&#39;iride per tenere la parete addominale, e utilizzare un altro paio di pinze dell&#39;iride per cercare i cuscinetti di grasso collegati alla epididimo e testicolo nella cavità peritoneale. Estrarre delicatamente il cuscinetto adiposo fino a che la testicolo viene esteriorizzato e l&#39;arteria testicolare e dell&#39;epididimo sono chiaramente visibili. Lavorare su una testicolo alla volta. </li><li> Posizionare un telino sterile sottile a base di schede in autoclave sotto il cuscinetto adiposo / testicolo per una migliore identificazione visiva (opzionale). Il drappo lavora anche per assorbire fluido. </li><li> Aggiungere una goccia di colorante Trypan Blue nella sospensione cellulare e accuratamente caricare la sospensione cellula nel polietitubazione Lene collegato ad una siringa 1 ml. Fissare la pipetta tirato nel tubo e delicatamente forzare la sospensione cellula nel pipetta applicando pressione alla siringa. </li><li> Identificare i condotti efferenti (che collegano le testicolo per l&#39;epididimo) e rimuovere delicatamente il tessuto grasso attorno ai dotti. Lavorare con cura, come i condotti e la membrana che li circondano sono trasparenti. </li><li> Inserire con cautela la pipetta in un condotto nel fascio di condotti efferenti, delicatamente infilare pochi mm verso il testicolo. </li><li> Mentre evitando muovendo il pipetta di iniezione, raggiungere per la siringa, delicatamente prema lo stantuffo della siringa per assicurare che la sospensione fluisce nel rete testis e seminiferi tubuli cominciano a riempire. </li><li> La velocità di iniezione e la portata di sospensione cellulare è regolato da pressione con il pollice. Evitare aumento improvviso di pressione; monitorare il movimento di sospensione in tubuli. </li><li> Interrompere l&#39;iniezione, quando quasi tutti i tubuli di superficie sono stati riempiti prima che il testicolo inizia a become ischemico. </li><li> Riportare il testicolo alla cavità addominale. Ripetere la procedura sul testicolo controlaterale. Suturare la parete addominale con suture in seta 6-0 e chiudere la pelle con clips metalliche ferita. Monitorare i topi su un pad riscaldamento fino al recupero completo. </li></ol> 5. Analisi dei testicoli beneficiarie <ol><li> Lasciare 2-4 mesi dopo il trapianto prima dell&#39;analisi. </li><li> Euthanize il mouse destinatario secondo la cura degli animali e le linee guida di utilizzo e raccogliere il testicolo e all&#39;epididimo in PBS. Quando un Lac-Z ceppo donatore transgenico è stato utilizzato, l&#39;epididimo può essere utilizzato come controllo colorazione positivo in quanto ha endogena beta-galattosidasi attività. </li><li> Per il rilevamento di cellule del donatore da Lac-Z colorazione, fissare il testicolo per 1-2 ora a 4 ° C in 4% paraformaldeide (PFA) in PBS. Sciacquare 3 x 30 min a temperatura ambiente in lacZ risciacquo tampone. </li><li> Incubare notte a 37 ° C in soluzione colorante lacZ. Testicolo può essere colorato nel suo insieme o dopo la dispersionei tubuli. </li><li> Fissare e memorizzare in 10% formalina tamponata neutra. Se le sezioni è necessario utilizzare rosso neutro veloce come anti-macchia. </li></ol> PARTE B. ectopica xenotrapianto in topi (Da Dobrinski e Rath 2008 15 e-Sosa Rodriguez et al. 2011 16). 1. Raccolta di tessuto di donatore <ol><li> Ottenere tessuto testicolare con la castrazione o la biopsia da un maschio donatore. </li><li> Testicolo di luogo in PBS o biopsie in terreno di coltura, mantenendo condizioni di sterilità. </li><li> Tenere il tessuto raccolti su ghiaccio e trasporto al laboratorio. </li></ol> 2. Preparazione di tessuto di donatore <ol><li> Eseguire in la cappa coltura tissutale per mantenere la sterilità. </li><li> Lavare ogni testicolo in gelide antibiotici PBS contenente due o tre volte prima di trasferire in una cultura piatto con PBS. Nel caso di biopsie, lavare frammenti testicolo due o tre volte con ghiaccio-freddo medi culturaum contenenti antibiotici, facendo girare a 150 g per 2 minuti e risospensione in acqua dolce ghiacciata terreno di coltura. </li><li> Per testicoli intatti, rimuovere tunica vaginale facendo un&#39;incisione lungo la superficie e estrudere testicolo. Togliere dal testicolo tutte le strutture annesse (spermatico cavo, epididimo, tessuto connettivo). Lavare i testicoli una volta in PBS freddo e il trasferimento in una cultura-piatto con PBS. </li><li> Rimuovere con cura la tunica albuginea del testicolo utilizzando una lama da bisturi e un paio di forbici. Se il testicolo è molto piccola, la tunica può essere rimosso dalla spremitura del tessuto testicolare fuori dalla tunica attraverso una piccola incisione fatto su uno estremità tenendo la tunica con un paio di pinze piccole sull&#39;altra estremità. </li><li> A seconda delle dimensioni del testicolo sia il tessuto testicolo intero può essere tagliato in piccoli pezzi di circa 1 - 2 mm 3 in dimensioni usando pinze curve e una lama bisturi, o pezzi grandi di tessuto testicolare può prima essere rimosso dal testicolo e poi tagliati in piccolipezzi. Tutto ciò dovrebbe essere fatto in ghiacciato terreno di coltura e in condizioni sterili in un piccolo piatto di coltura (60 x15 mm). </li><li> Trasferire i frammenti di tessuto preparate per ghiacciata terreno di coltura in piccoli piatti di cultura nel ghiaccio fino innesto. </li></ol> 3. La castrazione del destinatario del mouse <ol><li> Anestetizzare il mouse come sopra e preparare sterile campo chirurgico il taglio i capelli (non necessario in topi nudi), asciugandosi con il 70% di etanolo e la soluzione di Betadine. </li><li> Fare un 0,5 -1 cm ventrale incisione cutanea mediana per esporre la parete addominale. </li><li> Con attenzione esporre il testicolo, l&#39;arteria testicolare e dell&#39;epididimo, come descritto in Parta, 4,2-4,3. </li><li> Staccare la coda dei epidydimis dal gubernaculum per via smussa. </li><li> Legare l&#39;arteria testicolare, e dei vasi deferenti insieme con il vaso sanguigno con la seta, e la sezione delle strutture ligati tagliando tra il testicolo e la legatura. </li><li> Ripetere la procedura per il second testicolo. </li><li> Suturare la parete addominale con uno o due punti di sutura chirurgici. </li><li> Chiudere l&#39;incisione cutanea con uno o due clip Michel. </li></ol> 4. Ectopica xenotrapianto <ol><li> Posizionare il mouse in decubito ventrale e preparare un campo sterile chirurgico sulla sua schiena come sopra. </li><li> A seconda di quanti innesti sono di essere inserito (generalmente 4-8/mouse), rendono ~ 0.5 cm incisioni cutanee lunghe su ciascun lato della linea metà del retro del mouse. </li><li> Utilizzare pinze per tenere un bordo di incisione cutanea, e fare una cavità sottocutanea da prendere in giro a parte il tessuto connettivo con le forbici. </li><li> L&#39;utilizzo di un posto iris forcipe un pezzo di tessuto testicolare in profondità nella cavità sottocutanea, tenendo il bordo della incisione cutanea con un altro pinza iris. </li><li> Chiudere l&#39;incisione della pelle con una clip Michel e tenere il mouse sul tappetino di riscaldamento fino a quando non inizia a riprendersi dall&#39;anestesia. </li><li> Trasferire il mouse in una gabbia con isolamento supplementare e cover e il monitor fino a quando i topi sono completamente recuperati. </li></ol> 5. Raccolta di xenotrapianti testicolo per l&#39;analisi e la raccolta dello sperma <ol><li> Euthanize il mouse ospite secondo la cura degli animali e le linee guida d&#39;uso, e fare una incisione mediana della pelle sulla pelle running back dalla coda al collo e la pelle aperto. Questo espone i innesti che possono essere situate sia sul tessuto sottocutaneo o attaccate alla pelle. </li><li> Rimuovere con cautela gli innesti utilizzando una pinza coppia e un paio di forbici. </li><li> Numero record di innesti recuperato, le dimensioni e il peso dei singoli innesti. </li><li> Recuperare le vescicole seminali dall&#39;addome del mouse e registrare il loro peso come un&#39;indicazione di produzione di testosterone da parte del tessuto innestato. </li><li> Per l&#39;analisi istologica <ol class="lalpha"><li> Sospendere xenotrapianti in flaconcino campione contenente soluzione di Bouin (o fissativo altro) in un volume ~~~V 10x che del xenograft, e flaconcino etichetta adeguatamente. </li><li> Covarenotte in frigorifero seguita da lavaggio almeno 3 volte in etanolo 70% ad intervalli di 24 ore preferibilmente. </li><li> Procedere per l&#39;elaborazione e l&#39;incorporamento in paraffina. </li></ol></li><li> Per raccolta spermatozoi <ol class="lalpha"><li> Lavare xenotrapianti facendo girare giù a 300g per 1 min e risospensione in coltura terreno contenente antibiotici. </li><li> Tagliare innesti a pezzetti e tritate accuratamente con le pinze in un piatto di coltura contenente 3 - 5 ml di terreno di coltura. </li><li> Filtrare il tessuto tritato attraverso il filtro 40 micron cell. </li></ol></li></ol> PARTE C. rappresentativi Risultati 1. Trapianto di cellule germinali Se la sospensione di cellule del donatore trapiantato contiene cellule staminali spermatogoni che trasportano un marcatore genetico come il transgene LacZ, colonizzazione della SSC donatori nel testicolo destinatari possono essere visualizzati da X-gal colorazione come distintivi segmenti blu della seminiferous tubuli dopo 2-3 mesi post-trapianto 3 (Figura 2A). Una consolidata colonia dovrebbero avere un lungo tratto blu scuro di segmenti completamente riempite di due o più strati di cellule blu più vicini al centro, e regioni macchiati relativamente più deboli sia finisce dove una rete di singolo, coppia o piccoli gruppi di cellule blu è apparente 3 (Figura 2B). Una sezione trasversale della regione blu scuro tubulo seminifero dovrebbe rivelare una realtà consolidata e ben organizzata spermatogenesi con le cellule germinali blu ai vari stadi di differenziazione (Figura 2C). 2. Testicolo tessuto xenotrapianto La vitalità del tessuto testicolare dopo il trapianto è inversamente correlato con la fase di sviluppo del donatore, il miglior risultato si ottiene quando il tessuto da neonati maschi è usato, mentre il tessuto adulto mostra una forte tendenza a degenerare e morire 17-21. Generalmente, successo eterotrapianto diminuisce come tessuto donatore me subisceiosis della prima ondata della spermatogenesi. Tempo di pieno sviluppo del tessuto del donatore immature e la spermatogenesi completa è specie-specifico, e spesso un po &#39;più breve rispetto ai testicoli in situ. Il numero dei tubuli seminiferi con la spermatogenesi completa è variabile secondo le specie. Mentre in ovini, caprini e maiali che numero è maggiore di 50%, nei bovini e gatti è inferiore al 15% 14 (figura 3). <img alt="Figura 1" src="/files/ftp_upload/3545/3545fig1.jpg" /> Figura 1. Procedura di trapianto di cellule Germ. Place destinatari in decubito dorsale dopo l&#39;anestesia e fare un 0.4-pollici ~ un&#39;incisione addominale mediana (A). Esporre testicolo con la revoca della cuscinetto adiposo attaccato al epididimo e testicolo e posizionare un telino sterile sottile sotto il cuscinetto adiposo / testicolo per una migliore identificazione visiva (B). Posizionare il testicolo e all&#39;epididimo in modo che i condotti efferenti sepolti nel cuscinetto adiposo sono discernibili (C, D, E). Identità i condotti efferenti e rimuovere delicatamente il tessuto grasso attorno ai dotti. Un pezzo di carta colorata o di plastica può essere collocato sotto i condotti per una migliore visualizzazione (G). Rompere o macinare la punta della pipetta base alle dimensioni dei condotti e cella di sospensioni di carico nella pipetta (H). Inserire con cautela la pipetta in un condotto nel fascio di condotti efferenti, delicatamente infilare pochi mm verso il testicolo (la freccia in H indica la direzione di pipetta di iniezione e threading). Un testicolo con iniezione con successo nel tubuli seminiferi è mostrato (I). Ts: testicolo; Ep: epididimo. <img alt="Figura 2" src="/files/ftp_upload/3545/3545fig2.jpg" /> Figura 2. Risultati rappresentativi per il trapianto di cellule germinali A) Un testicolo iniettati colorati con X-gal. I segmenti blu dei tubuli seminiferi rappresentano le colonie stabilite da SSCS donatori LacZ transgenici (transgene). B) ingrandimento superiore di una colonia SSC a 3 mesi post-trapianto. The nella regione blu scuro al centro rappresenta spermatogenesi completa e le regioni celesti alle estremità rappresentano l&#39;estensione crescente della colonia. C) Una sezione istologica del buio blu tubulo seminifero (3 mesi post-trapianto) mostra spermatogenesi ben organizzata. Barre di scala in B e C sono 200 micron e 30 micron, rispettivamente. (Figure adattato dalla cella Annu Rev Dev Biol 2008;. 24:263-86 e Reprod Biol giugno 1999;. 60 (6) :1429-36). <img alt="Figura 3" src="/files/ftp_upload/3545/3545fig3.jpg" /> Figura 3. Xenotrapianto ectopica di tessuto testicolare immaturi da animali di grandi dimensioni in topi immunodeficienti. Frammenti di testicolo donatori immaturi (~ 1 mm 3) trapiantate sotto la pelle dorsale di topi immunodeficienti (A) sono in grado di sopravvivere e rispondere alle gonadotropine mouse. Come risultato, tessuto testicolare subisce uno sviluppo completo, compresa formazione di sperma fecondazione competente (B). Una volta xenotrapianti testicolo vengono raccolti (C)essi possono essere utilizzati per analisi o per ottenere spermatozoi (D) per ICSI (E) e produzione di embrioni (F). Bar pari a 50 micron (B, E e F) o 10 micron (D). (Modificato da Rodriguez-Sosa e Dobrinksi 2009 14).

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R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+testis+tissue+xenografting.">Recent developments in testis tissue xenografting.</a> Reproduction. 138 (2), 187-187 (2009).</li><li>Dobrinski, I., Rathi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+grafting+of+mammalian+testis+tissue+into+mouse+hosts.">Ectopic grafting of mammalian testis tissue into mouse hosts.</a> Methods in Molecular Biology. 139, 450-450 (2008).</li><li>Rodriguez-Sosa, J. R., Schlatt, S., Dobrinski, I., Seli, E., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+tissue+transplantation+for+fertility+preservation.">Testicular tissue transplantation for fertility preservation.</a> Fertility Preservation: Emerging Technologies and Clinical Applications. , 331-331 (2011).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+fate+and+seminiferous+tubule+development+in+bovine+testis+xenografts.">Germ cell fate and seminiferous tubule development in bovine testis xenografts.</a> Reproduction. 130 (6), 923-923 (2005).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+development+in+equine+testis+tissue+xenografted+into+mice.">Germ cell development in equine testis tissue xenografted into mice.</a> Reproduction. 131 (6), 1091-1091 (2006).</li><li>Kim, Y., Selvaraj, V., Pukazhenthi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+donor+age+on+success+of+spermatogenesis+in+feline+testis+xenografts.">Effect of donor age on success of spermatogenesis in feline testis xenografts.</a> Reproduction, Fertility, and Development. 19 (7), 869-869 (2007).</li><li>Arregui, L., Rathi, R., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenografting+of+adult+mammalian+testis+tissue.">Xenografting of adult mammalian testis tissue.</a> Animal Reproduction Science. 106 (1-2), 65-65 (2008).</li><li>Schlatt, S., Honaramooz, A., Ehmcke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+survival+of+adult+human+testicular+tissue+as+ectopic+xenograft.">Limited survival of adult human testicular tissue as ectopic xenograft.</a> Human Reproduction. 21 (2), 384-384 (2006).</li><li>Shinohara, T., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta1-+and+alpha6-integrin+are+surface+markers+on+mouse+spermatogonial+stem+cells.">beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5504-5504 (1999).</li><li>Kanatsu-Shinohara, M., Toyokuni, S., Shinohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD9+is+a+surface+marker+on+mouse+and+rat+male+germline+stem+cells.">CD9 is a surface marker on mouse and rat male germline stem cells.</a> Biology of Reproduction. 70 (1), 70-70 (2004).</li><li>Ryu, B. Y., Orwig, K. E., Oatley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+and+niche+microenvironment+on+spermatogonial+stem+cell+self-renewal.">Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal.</a> Stem Cells. 24 (6), 1505-1505 (2006).</li><li>Costoya, J. A., Hobbs, R. M., Barna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+Plzf+in+maintenance+of+spermatogonial+stem+cells.">Essential role of Plzf in maintenance of spermatogonial stem cells.</a> Nature Genetics. 36 (6), 653-653 (2004).</li><li>Morrow, C. M., Hostetler, C. E., Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV5+is+required+for+continuous+spermatogenesis+in+adult+mice+and+may+mediate+blood+testes+barrier+function+and+testicular+immune+privilege.">ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege.</a> Annals of the New York Academy of Sciences. 1120, 144-144 (2007).</li><li>Zeng, W., Snedaker, A. K., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+transplantation+of+porcine+testis+tissue.">Preservation and transplantation of porcine testis tissue.</a> Reproduction, Fertility and Development. 21 (3), 489-489 (2009).</li><li>Jahnukainen, K., Ehmcke, J., Hergenrother, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+maturation+of+primate+testis+by+xenografting+into+mice.">Accelerated maturation of primate testis by xenografting into mice.</a> Biology of Reproduction. 70 (5), 1500-1500 (2004).</li></ol>

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1. Trapianto di cellule germinali Cellule germinali trapianto fornisce l&#39;unico saggio funzionale per la conferma inequivocabile della presenza di cellule staminali spermatogoniali (SSCs) in una popolazione cellulare. Solo SSCs possono casa e colonizzare la nicchia SSC a livello della membrana basale e avviare donatore-derivato spermatogenesi. Trapianto di cellule germinali ha permesso di studiare e manipolare i CSS in un modo senza precedenti. La tecnica è stata utilizzata per la produzio...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Collagenasi (tipo IV) </td><td> Sigma </td><td> C5138 </td></tr><tr><td> Tripsina-EDTA </td><td> Invitrogen </td><td> 25200-056 </td></tr><tr><td> DNaseI </td><td> Sigma </td><td> DN25 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31053-028 </td></tr><tr><td> Trypan macchia blu </td><td> Invitrogen </td><td> 15250-061 </td></tr><tr><td> Nylon mesh cella </td><td> BD Biosciences </td><td> 352.340 (40 ≠ m) 352350 (70μm) </td></tr><tr><td> Busulfan </td><td> Sigma </td><td> B2635 </td></tr><tr><td> Thin-Wall capillari di vetro </td><td> Mondo Precision Instrument </td><td> TW 100-3 </td></tr><tr><td> BD intramedic tubo plyethylene (PE100) </td><td> BD </td><td> CA-63018-725 </td></tr><tr><td> Ethicon 6-0 Silk sutura </td><td> Ethicon </td><td> 706G </td></tr><tr><td> Ferita clip </td><td> BD </td><td> 427631 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> X-gal </td><td> Sigma </td><td> B4252 </td></tr><tr><td> Il ferrocianuro di potassio </td><td> Sigma </td><td> P9387 </td></tr><tr><td> Ferricianuro di potassio </td><td> Sigma </td><td> P3667 </td></tr><tr><td> cloruro di magnesio </td><td> Sigma </td><td> 208337 </td></tr><tr><td> sodio deossicolato </td><td> Sigma </td><td> D6750 </td></tr><tr><td> N, N-Dimetilformammide </td><td> Sigma </td><td> D4551 </td></tr><tr><td> Igepal CA-630 </td><td> Sigma </td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

Trapianto di cellule germinali è stato sviluppato dal Dr. Ralph Brinster e colleghi presso la University of Pennsylvania nel 1994 1,2. Queste rivoluzionarie studi hanno dimostrato che microiniezione di cellule germinali di topi donatori fertili nei tubuli seminiferi dei risultati infertili topi riceventi del donatore-derivato spermatogenesi e la produzione di spermatozoi da parte dell&#39;animale ricevente 2. L&#39;uso dei maschi donatori che trasportano il batterico β-galattosidasi identificazione del gene consentito di donatore-derivato spermatogenesi e trasmissione della aplotipo donatore alla prole dagli animali beneficiarie 1. Sorprendentemente, dopo il trapianto nel lume dei tubuli seminiferi, cellule germinali trapiantate riusciti a spostare dal vano luminale alla membrana interrato dove sono spermatogoni 3. E &#39;generalmente accettato che SSCs solo sono in grado di colonizzare la nicchia e ristabilire la spermatogenesi nel testicolo destinatario. Pertanto, cellule germinalitrapianto fornisce un approccio funzionale per studiare la nicchia delle cellule staminali nei testicoli e per caratterizzare le cellule staminali putativi spermatogoni. Per data, trapianto di cellule germinali viene utilizzato per delucidare di base biologia delle cellule staminali, per produrre animali transgenici attraverso manipolazione genetica di cellule germinali precedente al trapianto di 4,5, per studiare Sertoli cell-germinali interazione cellulare 6,7, SSC homing e colonizzazione 3, 8, così come SSC self-renewal e la differenziazione 9,10. Trapianto di cellule Germ è anche realizzabile in 11 specie di grandi dimensioni. In queste, le principali applicazioni sono la conservazione della fertilità, la diffusione della genetica nelle popolazioni animali d&#39;elite, e la generazione di animali transgenici come lo studio della spermatogenesi e la biologia SSC con questa tecnica è logisticamente più difficile e costoso rispetto ai roditori. Il trapianto di cellule germinali di specie di grandi dimensioni nei tubuli seminiferi dei risultati topi in colonizationi di cellule del donatore e l&#39;espansione spermatogoni, ma non nella loro piena differenziazione presumibilmente a causa di incompatibilità della cellula ricevente vano somatiche con le cellule germinali di specie filogeneticamente distanti 12. Un approccio alternativo è il trapianto di cellule germinali di specie di grandi dimensioni, insieme con il loro vano circostante somatica. Siamo la prima volta nel 2002, che piccoli frammenti di tessuto testicolare da maschi immaturi trapiantate sotto la pelle dorsale di topi immunodeficienti sono in grado di sopravvivere e sottoporsi a pieno sviluppo con la produzione di sperma fecondazione competente 13. Da allora tessuto testicolare xenotrapianto ha dimostrato di avere successo in molte specie ed è emerso come una valida alternativa per studiare lo sviluppo del testicolo e la spermatogenesi di grandi animali nei topi 14.

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

I protocolli per il trapianto di cellule germinali e xenotrapianto tessuto testicolare sono descritti. Trapianto di cellule germinali risultati in donatori-derived spermatogenesi nei testicoli dei destinatari e rappresenta un test funzionale ricostituzione per l&#39;identificazione delle cellule staminali spermatogoni (SSC). Tessuto testicolare xenotrapianto riproduce sviluppo dei testicoli e la spermatogenesi di specie diverse donatori in topi riceventi.

Germ trapianto di cellule e di tessuto testicolare xenotrapianto in topi

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

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JoVE Journal

Biology

29.6K Views.  University of Calgary. I protocolli per il trapianto di cellule germinali e xenotrapianto tessuto testicolare sono descritti. Trapianto di cellule germinali risultati in donatori-derived spermatogenesi nei testicoli dei destinatari e rappresenta un test funzionale ricostituzione per l'identificazione delle cellule staminali spermatogoni (SSC). Tessuto testicolare xenotrapianto riproduce sviluppo dei testicoli e la spermatogenesi di specie diverse donatori in topi riceventi.

Video: Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

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Biologia

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Il trapianto di midollo renale intero in Zebrafish adulti

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Il trapianto cardiaco eterotopico nel Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Trapianto di intestino piccolo Nei topi

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generazione di Human CD40 delle cellule B attivate

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tessuto Chimere mirate embrionali: Zebrafish gastrula trapianto di cellule

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Eterotopica e trapianto ortotopico tracheale in topi utilizzati come modelli per studiare lo sviluppo della malattia delle vie aeree obliterante

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planaria immobilizzazione, irradiazione parziale, e di tessuti

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Studiare proteolisi della ciclina B a livello di singola cellula in popolazioni di cellule intere

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...