Vogliamo riconoscere il supporto dell &#39;Università di Chicago Sezione di Urologia diretto dal Dr. Arieh Shalhav, e il direttore di Urologia Ricerca Dr. Carrie Rinker-Schaeffer. Vorremmo anche ringraziare il sostegno della Università di Chicago Comprehensive Cancer Center (UCCCC) guidato dal Dr. Michelle Le Beau. Abbiamo anche ringraziare l&#39;assistenza tecnica di esperti del Tissue struttura Human Resource Centro nucleo guidato dal Dr. Mark Lingen, e l&#39;assistenza di Leslie Martin e Mary Jo Fekete. Ringraziamo anche l&#39;immunoistochimica Nucleo Fondo gestito da Terri Li. Questo lavoro è stato finanziato dall&#39;Università di Chicago Dipartimento di Chirurgia, Sezione di Urologia; un American Cancer Society Institutional Research Grant (ACS-IRG, # IRG-58-004), un Cancer Center Support Grant (P30 CA14599); Il Brinson Fondazione, l&#39;Alvin Baum Fondo Famiglia; L&#39;Università di Chicago Board Cancer Research Foundation femminile; S. Kregel è supportato da un HHMI: Med-in-Grad Fellowsanca (56006772) e un Cancer Biology Training Grant (T32-CA09594). Infine, vorremmo ringraziare Robert Clark, il Dr. VenkateshKrishnan, e Nathan Stadick per la loro valutazione critica del manoscritto.

Questo studio è stato effettuato in stretta conformità con le raccomandazioni contenute nella Guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health. Il protocollo è stato approvato dalla University of Chicago Institutional Animal Care e del Comitato Usa (IACUC, numeri di protocollo 72066 e 72231). Tutto l&#39;intervento è stato eseguito in anestesia, e tutti gli sforzi sono stati fatti per ridurre al minimo la sofferenza. La linea di cellule staminali embrionali umane WA01 (H1; NIH-registrazione # 0043) è stata acquisita da WiCell (Madison, WI) e coltivate utilizzando il protocollo alimentatore indipendente con mTeSR1 supporti (Stem Cell Technologies, Vancouver, BC). Cellule ES sono stati utilizzati entro dieci passaggi di scongelamento. 1. Isolamento del seno urogenitale da mouse o Rat Embrioni <ol><li> Eutanasia di un mouse a tempo-gravidanza (17,5 giorni) o di ratto (18,5 giorni) per inalazione di CO 2 per 5 min. La morte è confermata dalla cessazione dei segni vitali nel ratto. Si interrompe quindi il collo umanamente. La ra è l&#39;eutanasia, a questo punto. Spruzzare il 70% di etanolo per sterilizzare la pelle del ventre e fianchi. Tagliare la pelle addominale e strati muscolari e poi tagliare l&#39;utero, separare gli embrioni di ratto dal sacco amniotico, e tagliare il cordone ombelicale. Trasferire gli embrioni in un tubo da 50 ml Falcon contenente soluzione salina bilanciata di ghiacciata Hank (HBSS) e tenere in ghiaccio. Feti sono sacrificati per decapitazione con le forbici chirurgiche. </li><li> Posizionare l&#39;embrione a 100 millimetri piatto di Petri. Bisecare l&#39;embrione con le forbici a livello dell&#39;ombelico (Figura 1A). Rimuovere lo stomaco e l&#39;intestino tenue. Rivelare le ovaie nell&#39;embrione femminile e testicolo nell&#39;embrione maschio (Figura 1B e 1C). (Le ovaie sono situate ai poli caudali dei reni. I testicoli sono approssimativamente al livello della vescica). In questa fase evolutiva, UGSM intercettare sia embrioni maschili e femminili sono in grado di indurre un lignaggio epiteliale prostatica in presenza di ospitantetestosterone. 4 Dopo questo punto il tempo di sviluppo, tuttavia, gemme prostatiche iniziare formando nel UGSM, e l&#39;isolamento di puro UGSM è molto impegnativo. 4 </li><li> Al microscopio dissezione, tagliare la parete addominale nella linea centrale con Vännäs forbici per esporre la vescica e la parete posteriore dell&#39;addome. Libero il tratto genitale superiore da allegati (in embrioni di sesso maschile, ha tagliato l&#39;uretere, Wolff condotto e Mülleriani condotto, in embrioni di sesso femminile, tagliare l&#39;uretere e Mülleriani condotto). Liberare la vescica dal cordone ombelicale. Tagliare l&#39;osso pubico e senza mezzi termini separato dalla parete anteriore del seno urogenitale con Dumont pinza sottile punta. Infine, separare la parete posteriore del seno urogenitale dal retto. Tagliare seno urogenitale con la vescica all&#39;estremità inferiore (dall&#39;uretra) e memorizzare il seno in blocco urogenitale (con la vescica) in ghiaccio HBSS freddo. </li></ol> 2. La separazione del Sinus Mesenchima urogenitale <ol><li&gt; Rimuovere la vescica dal seno urogenitale al microscopio di dissezione (Figura 1D). </li><li> Trasferire il seno urogenitale a un tubo falcon contenente 1% tripsina nel calcio (Ca 2 +) e magnesio (Mg 2 +) HBSS gratuiti. Posizionare il tubo in frigorifero a 4 ° C per 75 min. </li><li> Rimuovere la tripsina e neutralizzare tripsinizzazione con il 10% Fetal Bovin Serum (FBS) in DMEM/F12 medie. </li><li> Stuzzicare delicatamente il manicotto mesenchimale appiccicoso dal tubo epiteliale con un ago o Dumont pinza sottile punta (mantenendo il tubo epiteliale intatta riduce le possibilità di contaminazione delle cellule epiteliali del mesenchima, questo può causare ghiandole roditori formando insieme al tuo cellule staminali di derivazione umana epitelio ghiandolare) (Figura 1D). 12 </li><li> Trasferire UGSM in un nuovo tubo Falcon contenente DMEM/F12 medio + 10% FBS + 1% di penicillina / streptomicina (Pen / Strep; 0,5 mg / ml) + Aminoacidi non essenziali 1% (NEAA) più 1nM R1881. Place il tubo in un incubatore a 37 ° C con 5% di CO 2 durante la notte. </li><li> Centrifugare il tessuto a 200g e scartare il surnatante. Lavare con tampone fosfato salino (PBS) (non disturbare il precipitato di tessuto). </li><li> Aggiungere 0,2% collagenasi (in DMEM/F12) per ri-sospendere il tessuto. Incubare a 37 ° C con il dolce dondolio per 1 ora. </li><li> Vigorosamente vortice il tessuto digestione tre volte fino a diventare miscela cella singola omogeneo e aggiungere DMEM/F12 media + 10% FBS + 1% P / S + 1% NEAA + 1 nM R1881 per neutralizzare la collagenasi. </li><li> Centrifugare a 200 xg e poi lavare da ri-sospensione delle cellule UGSM in HBSS, e ripetere tre volte per lavare completamente il UGSM. Infine sospendere cellule mesenchimali in DMEM/F12 e contare le cellule utilizzando un emocitometro o un dispositivo di conteggio delle cellule. </li><li> Dissociare le cellule hES colta protocollo utilizzando alimentatore-Free con mTeSR1 supporti (Stem Cell Technologies, Vancouver, BC) utilizzando un digestione Accutase (Millipore, Billerica, MA). Lavare le cellule HESdurante la fase logaritmica della loro crescita in caldi DMEM/F12 supporti, quindi aggiungere tiepida soluzione Accutase 1x, e incubare a 37 ° C per 15-20 minuti fino a quando le cellule singole sono uniformemente dissociata dalla colonie. Aggiungere una quantità uguale di 1x definito inibitore della tripsina (Invitrogen, Grand Island, NY) per neutralizzare il Accutase e pipettare delicatamente su e giù per mescolare e rompere grumi di cellule. Cellule Pipettare in una provetta da centrifuga e centrifugare per 3 minuti a 200 xg, e risospendere il pellet cellulare in DMEM/F12 media. Centrifugare tubi per 3 min a 200 xg di nuovo e rimuovere il surnatante, quindi risospendere in HBSS freddi o DMEM/F12 al volume desiderato. </li><li> Aggiungi cellule hES con 1:2.5 rapporto di HES / cellule mesenchimali. 3 Questa sarà una composizione cellulare del 1E 5 celle UGSM con 4E 4 hESCs per iniezione 10 ml (un singolo ratto UGS tipicamente produce circa 2E 6 cellule mesenchimali). Centrifugare a 200 xg per 5 minuti e poi scartare il surnatante. Risospendere con il 75% Matrigel (BD Biosciences,San Jose, CA, miscelato con il 25% HBSS freddo per una più facile gestione) e posizionare il tubo in ghiaccio per iniezione sotto-capsula renale. </li></ol> 3. Trapianto di tessuto ricombinante Sotto la capsula renale <ol><li> Preparare una area chirurgica sterile. </li><li> Anestetizzare un maschio atimico nudo mouse con ketamina / xylazina ip; utilizzare una miscela fresca che contiene un rapporto di ketamina 01:01:08: xylazina: salina. Il dosaggio sarà di 100 mg / kg di ketamina e 2,5 mg / kg Xylazina. Gli animali che non rispondono ad un dito del piede-pizzico dovrebbero essere pienamente sotto per 20-30 min. </li><li> Chirurgicamente preparare il mouse con la rasatura del sito chirurgico (se non si utilizza un mouse nudo), Risanamento con tre alternati salviette di alcol al 70% seguita da soluzione Betadine e l&#39;applicazione di un telo chirurgico. </li><li> Mettere umidità collirio (Altalube occhio unguento) per gli occhi del mouse per evitare l&#39;essiccazione durante l&#39;anestesia. </li><li> Durante l&#39;intervento chirurgico, la frequenza respiratoria e la temperatura corporea dei topi sono monitorati. I tassi di respirazione should essere costante e mantenuta all&#39;interno di 40-90 atti / min. Temperatura corporea viene monitorata con osservazione del colore delle mucose e le suole rosa dei piedi. </li><li> Fare un cm lunga incisione cutanea 1.0 longitudinale sul dorso. Continuare a tagliare la parete addominale in una incisione longitudinale 0,5 cm. </li><li> Premere delicatamente il rene fuori del ventre e mantenere umido superficie rene usando gocce di soluzione salina sterile. </li><li> Delicatamente forare la capsula renale, utilizzando un ago di siringa vuota calibro 27. Una puntura molto superficiale è sufficiente. Questo sarà dove si inserisce l&#39;ago senza punta per iniettare la miscela cellulare. </li><li> Riempire una siringa con ago senza punta 28-gauge con 10 ml di miscela tuo cellulare. Inserire lentamente il sito di puntura e penetrare il parenchima renale di raggiungere una zona sotto la capsula renale. Iniettare 10 pl della miscela cellulare per formare un bolo sul rene sotto la capsula renale. Ritiro l&#39;ago. </li><li> Riportare il rene alla Abdocavità terminale. Fissare lo strato muscolare del peritoneo con punti di sutura in nylon 4-0. </li><li> Chiudere la pelle con o sutura o fiocco. </li><li> Pulire il sangue dalla pelle con soluzione salina sterile. </li></ol> 4. Analgesia post-operatoria e di monitoraggio <ol><li> Iniettare l&#39;animale con buprenorfina (0,1 mg / kg ogni 12 ore) o ketoprofene (3-5 mg / kg in soluzione salina ogni 12 ore) per la gestione del dolore post-operatorio, e 1 ml di 37 ° C ip soluzione fisiologica per mantenere l&#39;animale caldo e prevenire la disidratazione. </li><li> Mettere l&#39;animale in una gabbia pulita e posizionarlo su una piastra elettrica mite. </li><li> Respingere gli animali per camera animale una volta che riprendano conoscenza e si muovono all&#39;interno della gabbia. </li><li> Osservare l&#39;animale al giorno per segni inattesi di malattie e infezioni per i successivi tre giorni. I topi sono monitorati con osservazione delle attività in gabbie tra cui cibo accesso e l&#39;acqua 1 giorno post-operatorio. Se i topi mostrano un minor numero di attività, ketoprofene è intraperitoneale annuncioministrato ancora una volta al giorno. </li><li> Rimuovere i punti della pelle o suture due settimane post-operatorie. </li><li> Tessuti xenotrapianto può essere raccolto a partire da 8 settimane dopo l&#39;intervento chirurgico. Xenotrapianti possono essere esposte in vivo utilizzando ultrasuoni piccoli animali (Figura 2A), e tessuti fissati possono essere sezionati e colorati per varie proteine ​​usando immunoistochimica (Figura 3). </li></ol>

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Gli autori non hanno alcun conflitto di interessi con il lavoro presentato.

Ricombinazione tessuto usando UGSM è una tecnica estremamente utile indagare lo sviluppo della prostata e gli eventi molecolari che portano al cancro della prostata iniziazione. Il potenziale induttivo di UGSM è stata utilizzata per numerose applicazioni nel campo della ricerca della prostata; questi includono miglioramento tumore prendere di linee di cellule della prostata e tumori, lo studio delle interazioni stroma-epiteliali, e la formazione di cross-specie ricombinanti prostata 7,17-20 Una corretta pre...

C&#39;è una grande necessità di migliori sistemi di modelli umani di cancro alla prostata. In particolare, i pertinenti sistemi modello umano del normale, i tessuti della prostata non-maligne che possono essere geneticamente manipolate per discernere direttamente il ruolo di specifici geni nell&#39;insorgenza del cancro alla prostata sarebbero incredibilmente informativo. L&#39;avvento dell&#39;era genomica ha individuato numerosi geni che possono avere un ruolo nella formazione del cancro. La mancanza di sistemi sperimentali modello umano, però, compromette gravemente la nostra capacità di testare e caratterizzare funzionalmente candidati cancro alla prostata geni di suscettibilità. Un sistema modello ideale dovrebbe facilitare le analisi funzionali rapidi e più rapido di geni di suscettibilità al cancro in combinazione con opportuni sistemi modello di roditori transgenici. Inoltre, un tale sistema modello umano consentirebbe caratterizzazione molecolare dei meccanismi di segnalazione di carcinogenesi prostatica verso la scoperta e la convalida di nuove terapie perprevenire la formazione del cancro alla prostata. Cellule staminali embrionali umane (hESC) sono in grado di formare tessuti prostatici umani come xenotrapianti. Nel 2006 Taylor, et al. Riferito che hESCs possono essere indotte a formare epiteli prostatica in vivo quando ri-unita roditore seno urogenitale mesenchima (UGSM) entro un periodo di 8-12 settimane. 1 Questi studi erano basati su precedenti lavori da il laboratorio Cunha mostrando che roditore UGSM embrionale può promuovere la differenziazione prostatica delle cellule staminali embrionali e le popolazioni di cellule epiteliali in vivo. 2,3 La prostata si sviluppa da un Anlagen embrionale chiamato il seno urogenitale (UGS), e prima di giorno embrionale 17 (topo E17 , giorno E18 nel ratto) le UGS possono essere rimossi e fisicamente divisi in epitelio (UGSE) e mesenchima (UGSM) 4 Questo approccio ricombinazione tessuto ha migliorato significativamente la nostra comprensione dello sviluppo della prostata e carcinogenesi, particolare.fattore di crescita ly e ormonali vie di segnalazione e le relazioni molecolari tra prostata stroma e l&#39;epitelio. 5-8 Questo metodo comporta la combinazione ex vivo di UGSM con staminali o cellule epiteliali della stessa o di diversa specie e queste ricombinanti cellulare / tissutale vengono impiantati e coltivati e xenotrapianti all&#39;interno ospitante topo. 4,9 Dopo un periodo di crescita in vivo, l&#39;impianto contiene prostata epiteliali strutture ghiandolari incorporate nel tessuto stromale. Ulteriori colorazione può essere condotta per determinare se tali strutture sono veramente prostatica e di origine umana. 10,11

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Hank's Balanced Salt Solution (HBSS)</td>
 <td>GIBCO</td>
 <td>14170</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>GIBCO</td>
 <td>11330</td>
 <td></td>
 </tr>
 <tr>
 <td>R1881</td>
 <td>Sigma</td>
 <td>965-93-5</td>
 <td>Mix to 1 ug/ml in Ethanol (1,000x stock)</td>
 </tr>
 <tr>
 <td>NEAA</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen-Strep Solution</td>
 <td>GIBCO</td>
 <td>15070</td>
 <td>100x stock</td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD Biosciences</td>
 <td>354230</td>
 <td></td>
 </tr>
 <tr>
 <td>KETASET (ketamine hydrochloride) </td>
 <td>Fort Dodge Animal Health</td>
 <td>NDC 0856-2013-01</td>
 <td>100 mg/ml; dilute 1:10 in sterile saline </td>
 </tr>
 <tr>
 <td>AnaSed (xylazine) </td>
 <td>VET-A-MIX, Inc.</td>
 <td>NADA 139-236</td>
 <td>20 mg/ml; dilute 1:10 in sterile saline</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>BD Biosciences</td>
 <td>215240</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma</td>
 <td>C2014</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketoprofen </td>
 <td>Fort Dodge</td>
 <td>NDC 0856-4396-01</td>
 <td>100 mg/ml; dilute 1:1,000 in sterile saline</td>
 </tr>
 <tr>
 <td>Altalube eye ointment </td>
 <td>Altaire Pharmaceuticals, Inc.</td>
 <td>NLC 56641-19850</td>
 <td></td>
 </tr>
 <tr>
 <td>Leica MZ16 F Stereomicroscope</td>
 <td>Leica</td>
 <td></td>
 <td>Any good dissecting scope can be used.</td>
 </tr>
 <tr>
 <td>Vannas spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15001-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>84855</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)</td>
 <td>Hamilton</td>
 <td>7803-02 </td>
 <td>Custom Needle </td>
 </tr>
 <tr>
 <td>Ethanol Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-62 </td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Gauze Pads</td>
 <td>Fisher Scientific</td>
 <td>22-415-469 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethicon Vicryl Suture (4-0 FS-2)</td>
 <td>MedVet International</td>
 <td>J392H </td>
 <td>Needle-in, dissolvable suture </td>
 </tr>
 <tr>
 <td>Autoclip 9 mm Wound Clips</td>
 <td>Becton Dickenson</td>
 <td>427631</td>
 <td></td>
 </tr>
 <tr>
 <td>PVP Iodine Prep Pads</td>
 <td>Fisher Scientific</td>
 <td>06-669-98 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissector scissor with blunt end</td>
 <td>Fine Science Tools</td>
 <td>14072-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont fine tip forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder with Scissor</td>
 <td>Fine Science Tools</td>
 <td>12002-14</td>
 <td></td>
 </tr>
 </tbody>
</table>

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

I progressi nella ricerca sul cancro alla prostata è fortemente limitata dalla disponibilità di sistemi modello di derivazione umana e l&#39;ormone-naïve, che limitano la nostra capacità di comprendere gli eventi genetici e molecolari alla base delle malattie della prostata iniziazione. Verso lo sviluppo di migliori sistemi modello per lo studio di carcinogenesi della prostata umana, noi e altri hanno approfittato della unico potenziale induttivo pro-prostatica di embrionale roditore stroma della prostata, del seno urogenitale chiamato mesenchima (UGSM). Quando ricombinato con alcune popolazioni di cellule pluripotenti come le cellule staminali embrionali, UGSM induce la formazione di umana normale della prostata epiteli in maniera testosterone-dipendente. Tale sistema modello umano può essere usato per studiare e sperimentalmente testare la capacità del candidato cancro alla prostata suscettibilità geni a un ritmo accelerato rispetto ai tipici studi transgenici roditori. Dal momento che le cellule staminali embrionali umane (hESC) possono essere geneticamente modificati nella cultura Using espressione genica inducibile o siRNA vettori di knock-down prima della ricombinazione dei tessuti, tale modello facilita la verifica delle conseguenze funzionali di geni, o combinazioni di geni, che si pensa di promuovere o prevenire la carcinogenesi. La tecnica di isolare popolazioni pure di cellule UGSM, però, è impegnativo e di apprendimento spesso richiede una persona con esperienza precedente di insegnare personalmente. Inoltre, l&#39;inoculazione di miscele di cellule sotto la capsula renale di un ospite immunocompromesso può essere tecnicamente impegnativo. Qui delineare ed illustrare il corretto isolamento dei UGSM da embrioni di roditori e capsula renale impianto di miscele di tessuto per formare epitelio prostatico umano. Tale approccio, nella sua fase attuale, richiede in vivo xenotrapianto di cellule staminali embrionali; applicazioni future potrebbero potenzialmente includere nella formazione ghiandola vitro o l&#39;uso di pluripotenti indotte popolazioni di cellule staminali (iPSCs).

Sulla base del rapporto emozionante da Taylor, et al., Il nostro laboratorio ha messo a punto un protocollo engrafting utilizzando l&#39;H1 comunemente usato (NIH-designato WA01, geneticamente maschio) linea di cellule staminali embrionali umane. 1 Questa linea è stato rigorosamente testato per il controllo di qualità e è cariotipo normale 13 Quando coltivate in modo appropriato, le cellule HES possono essere mantenute, ampliato, e crioconservati in uno stato indifferenziato e pluripote...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Per svelare sono disperatamente necessari dei primi meccanismi molecolari alla base della prostata cancro iniziazione, il romanzo e sistemi modello umane innovative e approcci. Il potenziale di pre-prostatica seno urogenitale mesenchima (UGSM) per indurre pluripotenti popolazioni di cellule staminali di formare epitelio prostatico umano è un potente strumento sperimentale nella ricerca della prostata.

Formazione di epitelio prostatico umano utilizzando tessuti ricombinazione dei roditori urogenitale Mesenchima Sinus e cellule staminali umane

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

formation-human-prostate-epithelium-using-tissue-recombination-rodent

Research

JoVE Journal

Medicine

13.6K Views.  University of Chicago. Per svelare sono disperatamente necessari dei primi meccanismi molecolari alla base della prostata cancro iniziazione, il romanzo e sistemi modello umane innovative e approcci. Il potenziale di pre-prostatica seno urogenitale mesenchima (UGSM) per indurre pluripotenti popolazioni di cellule staminali di formare epitelio prostatico umano è un potente strumento sperimentale nella ricerca della prostata.

Video: Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Trapianto di cellule staminali in un In vitro Simulato ischemia / riperfusione Modello

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Ricerca

Medicina

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Iniezione sottoretinico di vettori di terapia genica e cellule staminali nel Eye mouse

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

La valutazione delle proprietà sulle cellule staminali in cellule di carcinoma ovarico umano Utilizzo multi e single cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-capture Microdissezione di Human prostatica Epitelio per l&#39;analisi di RNA

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pre-clinica ortotopico Modello murino di Human cancro alla prostata

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Generazione di un umanizzato mouse Fegato Utilizzando epatici umani cellule staminali

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Valutazione della funzione microvascolare di tessuto adiposo umano mediante videomicroscopia

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...