Queremos agradecer el apoyo de la Universidad de Chicago Sección de Urología dirigido por el Dr. Arieh Shalhav, y el Director de Investigación Urológica Dr. Carrie Rinker-Schaeffer. También nos gustaría agradecer el apoyo de la Universidad del Centro Integral del Cáncer de Chicago (UCCCC), dirigido por la Dra. Michelle Le Beau. También dar las gracias a la asistencia técnica de expertos de la instalación Human Tissue Resource Center básico dirigido por el Dr. Mark Lingen, y la ayuda de Leslie Martin y Mary Jo Fekete. También agradecemos a la facilidad de la base inmunohistoquímica dirigido por Terri Li. Este trabajo fue financiado por la Universidad del Departamento de Cirugía de Chicago, la Sección de Urología, la Sociedad Americana del Cáncer Beca de Investigación Institucional (ACS-IRG, # IRG-58-004), un Centro del Cáncer de Apoyo Grant (P30 CA14599); El Brinson Fundación, el Fondo Alvin Baum Familia, la Universidad de Chicago Board Cancer Research Foundation de la Mujer; S. Kregel el apoyo de un HHMI: Med-en-Grad becarioscadera (56006772) y Biología del Cáncer Training Grant (T32-CA09594). Por último, nos gustaría dar las gracias a Robert Clark, Dr. VenkateshKrishnan y Nathan Stadick para su evaluación crítica del manuscrito.

Este estudio se llevó a cabo en estricta conformidad con las recomendaciones de la Guía para el Cuidado y Uso de Animales de Laboratorio del Instituto Nacional de Salud. El protocolo fue aprobado por la Universidad de Chicago Institucional Cuidado de Animales y el empleo Comisión (IACUC, números de protocolo 72066 y 72231). Toda la cirugía se realizó bajo anestesia, y se hicieron todos los esfuerzos para minimizar el sufrimiento. La madre de embriones humanos línea celular WA01 (H1; NIH-registro # 0043) fue adquirido de WiCell (Madison, WI) y se cultivaron usando el protocolo de alimentación independiente con mTeSR1 medios (Stem Cell Technologies, Vancouver, BC). Las células ES se utilizaron dentro de los diez pasajes de descongelación. 1. El aislamiento del seno urogenital de ratón o rata embriones <ol><li> La eutanasia a un ratón temporizada embarazadas (17,5 días) o la rata (18,5 días) por inhalación de CO 2 durante 5 min. La muerte se confirma por el cese de los signos vitales en la rata. A continuación, cortar la cabeza humana. El ra es la eutanasia en este punto. Pulverizar etanol al 70% para esterilizar la piel del abdomen y los flancos. Cortar la piel del abdomen y las capas de músculo y luego se corta el útero, separe los embriones de rata de la bolsa amniótica, y cortar el cordón umbilical. La transferencia de los embriones en un tubo Falcon de 50 ml que contenía solución salina equilibrada de Hank enfriada en hielo (HBSS) y mantener en hielo. Los fetos son sacrificados por decapitación con tijeras quirúrgicas. </li><li> Coloque el embrión en 100 mm de placa de Petri. Biseccionar el embrión con las tijeras en el nivel del ombligo (Figura 1A). Retire el estómago y el intestino delgado. Revelar los ovarios en el embrión femenino y testículos en el embrión masculino (Figura 1B y 1C). (Los ovarios están ubicados en los polos caudal de los riñones. Los testículos son aproximadamente al nivel de la vejiga). En esta etapa de desarrollo, aislado UGSM de ambos embriones macho y hembra son capaces de inducir un linaje epitelial prostática en presencia de anfitrióntestosterona. 4 Después de este punto en el tiempo de desarrollo, sin embargo, las yemas prostáticas comenzar a formar en el UGSM, y el aislamiento de UGSM puro es muy difícil. 4 </li><li> Bajo el microscopio de disección, corte de la pared abdominal en la línea media con unas tijeras Vannas para exponer la vejiga y de la pared posterior del abdomen. Libre el tracto genital superior de los apegos (embriones masculinos, cortó el uréter, el conducto de Wolff y de Müller conducto, en los embriones femeninos, corte el uréter y conductos de Müller). Libera la vejiga del cordón umbilical. Cortar hueso púbico y sin rodeos separada de la pared anterior del seno urogenital con pinzas de punta fina Dumont. Por último, separar la pared posterior del seno urogenital del recto. Cortar el seno urogenital con la vejiga en el extremo inferior (de la uretra) y almacenar el seno urogenital en bloque (con la vejiga) en HBSS frío de hielo. </li></ol> 2. La separación del mesénquima seno urogenital <ol><li&gt; Quite la vejiga del seno urogenital bajo el microscopio de disección (Figura 1D). </li><li> Transferir el seno urogenital a un tubo Falcon que contiene 1% de tripsina en calcio (Ca 2 +) y magnesio (Mg 2 +) HBSS libre. Colocar el tubo en un refrigerador a 4 º C durante 75 min. </li><li> Retire la tripsina y neutralizar tripsinización con 10% de suero de Bovin fetal (FBS) en medio DMEM/F12. </li><li> El pelo con cuidado el manguito mesenquimal pegajosa del tubo epitelial con una aguja o Dumont fórceps de punta fina (manteniendo el tubo epitelial intacta reduce las posibilidades de contaminación de células epiteliales del mesénquima, lo que puede resultar en la formación de las glándulas de roedores, junto con su tallo derivado de células humana epitelio glandular) (Figura 1D). 12 </li><li> Transferencia UGSM en un nuevo tubo Falcon que contiene medio DMEM/F12 + 10% de FBS + 1% de penicilina / estreptomicina (Pen / Strep; 0,5 mg / ml) + 1% de aminoácidos no esenciales (NEAA), además de 1 nM R1881. Plas el tubo en una incubadora a 37 ° C con 5% de CO 2 durante la noche. </li><li> Se centrifuga el tejido a 200 xg y descartar el sobrenadante. Lavar con tampón fosfato salino (PBS) (no perturbar el sedimento de tejido). </li><li> Añadir 0,2% de colagenasa (en DMEM/F12) para volver a suspender el tejido. Incubar en 37 ° C con agitación suave durante 1 hora. </li><li> Vórtice vigorosamente el tejido digestión tres veces hasta que se convierte mezcla homogénea de una sola célula y añadir medio DMEM/F12 + 10% de FBS + 1% P / S + 1% de NEAA + 1 nM R1881 para neutralizar la colagenasa. </li><li> Centrifugar a 200 xg y lavar por la re-suspensión de las células UGSM en HBSS, y repetir tres veces para lavar totalmente el UGSM. Finalmente suspender las células mesenquimales en DMEM/F12 y contar las células usando un hemocitómetro o un dispositivo de recuento de células. </li><li> Disociar HES células cultivadas usando el protocolo de enlace de conexión con mTeSR1 medios (Stem Cell Technologies, Vancouver, BC) utilizando una digestión Accutase (Millipore, Billerica, MA). Lavar las células hESdurante la fase logarítmica de su crecimiento en medio DMEM/F12 cálidos, a continuación, agregar solución Accutase 1x caliente, y se incuba a 37 ° C durante 15-20 minutos hasta que las células individuales son uniformemente disociada de colonias. Añadir la misma cantidad de 1x definido inhibidor de tripsina (Invitrogen, Grand Island, NY) para neutralizar el Accutase y pipetear suavemente hacia arriba y hacia abajo para mezclar y romper los grumos de células. Células pipeta en un tubo de centrífuga y centrifugar durante 3 minutos a 200 xg, y volver a suspender el sedimento celular en medio DMEM/F12. Centrifugar tubo durante 3 minutos a 200 xg una y separar el sobrenadante y vuelva a suspender en HBSS frío o DMEM/F12 al volumen deseado. </li><li> Añadir HES células con relación 1:2,5 de HES / células mesenquimales. 3 Esta será una composición celular de las células 1E 5 UGSM con 4E 4 hESCs por inyección de 10 ml (una sola rata UGS típicamente rendimientos de alrededor de 6 células mesenquimales 2E). Centrifugar a 200 xg durante 5 minutos y después se desecha el sobrenadante. Vuelva a suspender el 75% Matrigel (BD Biosciences,San Jose, CA, mezclado con 25% HBSS frío para un manejo más fácil) y colocar el tubo en hielo durante la inyección cápsula sub-renal. </li></ol> 3. Trasplante de tejido recombinante Por debajo de la cápsula renal <ol><li> Prepare un área quirúrgica estéril. </li><li> Anestesie un ratón nude atímicos macho con ip ketamina / xilazina, el uso de una mezcla fresca que contiene una proporción de 01:01:08 ketamina: xilazina: solución salina. La dosificación será 100 mg / kg de ketamina y 2,5 mg / kg de xilazina. Los animales que no responden a un dedo del pie para los dedos deben estar totalmente bajo durante 20-30 min. </li><li> Quirúrgicamente preparar el ratón por el afeitado de la zona quirúrgica (si no está usando un ratón desnudo), la limpieza con tres paños alternados de alcohol al 70% seguido de solución de betadine y la aplicación de un paño quirúrgico. </li><li> Ponga la humedad colirio (pomada para los ojos Altalube) a los ojos del ratón para evitar que se seque durante la anestesia. </li><li> Durante la cirugía, la tasa de respiración y la temperatura corporal central de los ratones son monitoreados. Las tasas de respiración should ser constante y mantenerse dentro de 40 a 90 respiraciones / min. La temperatura corporal central se controla con la observación del color de las membranas mucosas y las plantas de los pies de color rosa. </li><li> Hacer un cm de largo incisión longitudinal 1.0 en la parte posterior. Continuar para cortar la pared abdominal en una incisión longitudinal de 0,5 cm. </li><li> Apriete suavemente el riñón fuera del abdomen y mantener la humedad de la superficie del riñón usando gotas de solución salina estéril. </li><li> Suavemente perforar la cápsula renal con una aguja de jeringa de calibre 27 vacía. Una punción muy superficial es suficiente. Este será el lugar donde se inserta la aguja roma para inyectar la mezcla celular. </li><li> Llene una jeringa con aguja roma de calibre 28 con 10 l de la mezcla de células. Inserte lentamente la zona de punción y penetrar en el parénquima renal para llegar a un área por debajo de la cápsula renal. Inyectar 10 l de la mezcla celular para formar un bolo en el riñón por debajo de la cápsula renal. Retirada de la aguja. </li><li> Devuelva el riñón a la abdocavidad del terminal. Asegure la capa muscular del peritoneo con sutura de nylon 4-0. </li><li> Cierre de la piel, ya sea con suturas o grapas. </li><li> Limpiar la sangre de la piel usando solución salina estéril. </li></ol> 4. Analgesia postoperatoria y Monitoreo <ol><li> Inyectar el animal con buprenorfina (0,1 mg / kg cada 12 h) o ketoprofeno (3-5 mg / kg en solución salina cada 12 h) para controlar el dolor post-operatorio, y 1 ml de 37 ° C IP solución salina para mantener caliente el animal y prevenir la deshidratación. </li><li> Coloque el animal en una jaula limpia y colocarlo en una almohadilla eléctrica leve. </li><li> Enviar a los animales de vuelta a la sala de los animales una vez que recobren el conocimiento y se mueven dentro de la jaula. </li><li> Observe que el animal diariamente para detectar signos inesperados de enfermedades e infecciones de los tres días siguientes. Los ratones se controlan con la observación de las actividades en jaulas incluyendo comida acceso y agua de 1 día después de la operación. Si los ratones muestran menos actividades, ketoprofeno es intraperitoneal administró de nuevo una vez al día. </li><li> Retire las grapas o suturas cutáneas 2 semanas después de la cirugía. </li><li> Tejidos xenoinjertos se pueden cosechar a las 8 semanas después de la cirugía. Los xenoinjertos se pueden obtener imágenes in vivo utilizando ultrasonido de pequeños animales (Figura 2A), y los tejidos fijos pueden ser seccionadas y teñidas para diversas proteínas mediante inmunohistoquímica (Figura 3). </li></ol>

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Los autores no tienen ningún conflicto de intereses con el trabajo presentado.

Tejido por medio de recombinación UGSM es una técnica muy útil para investigar el desarrollo de la próstata y los eventos moleculares que conducen a la iniciación del cáncer de próstata. El potencial inductivo de UGSM se ha utilizado en numerosas aplicaciones en la investigación de próstata, que incluyen la mejora del tumor toma de líneas celulares de próstata y tumores, el estudio de las interacciones epiteliales del estroma-, y la formación de recombinantes de próstata de especies cruzadas 7,17-20</s...

Hay una tremenda necesidad de mejores sistemas de modelos humanos de cáncer de próstata. En particular, sistemas de modelos humanos pertinentes de tejidos de próstata normales, no malignas que pueden ser manipuladas genéticamente para discernir directamente el papel de genes específicos en la iniciación del cáncer de próstata serían muy informativo. El advenimiento de la era genómica ha identificado numerosos genes que pueden tener un papel en la formación del cáncer. La falta de sistemas de modelos experimentales humanos, sin embargo, perjudica gravemente nuestra capacidad para probar y caracterizar funcionalmente genes candidatos de susceptibilidad al cáncer de próstata. Un sistema modelo ideal sería facilitar los análisis funcionales rápidos y más rápida de genes de susceptibilidad al cáncer en combinación con sistemas de modelos de roedores transgénicos adecuados. Por otra parte, un sistema modelo humano tal permitiría la caracterización molecular de los mecanismos de señalización de la carcinogénesis de próstata hacia el descubrimiento y validación de nuevas terapias paraevitar la formación de cáncer de próstata. Células madre embrionarias humanas (hESCs) son capaces de formar los tejidos prostáticos humanos como xenoinjertos. En 2006 Taylor, et al. Informó que hESCs pueden ser inducidas a formar epitelios de próstata in vivo cuando se re-combinado con roedores seno urogenital mesénquima (UGSM) dentro de un período de tiempo de 8-12 semanas. 1 Estos estudios se basaban en el trabajo previo de el laboratorio Cunha que muestra que roedores UGSM embrionario puede promover la diferenciación de las células madre de próstata y poblaciones de células epiteliales embrionarias in vivo. 2,3 La próstata se desarrolla a partir de un Anlagen embrionario denominado el seno urogenital (UGS), y antes de que el día embrionario 17 (E17 ratón ; día E18 en la rata) el UGS se pueden quitar y divididos físicamente en el epitelio (UGSE) y el mesénquima (UGSM) 4 Este enfoque recombinación tejido ha mejorado significativamente nuestra comprensión del desarrollo y la carcinogénesis de próstata, en particular.factor de crecimiento de LY y vías de señalización hormonales y las relaciones moleculares entre la próstata estroma y el epitelio. 5-8 Este método implica la combinación ex vivo de UGSM con tallo o células epiteliales de las mismas o distintas especies y estos recombinantes celulares / tejido se implantan y se cultivaron y xenoinjertos en ratón huésped. 4,9 Después de un período de crecimiento in vivo, el implante contiene próstata estructuras glandulares epiteliales incrustados en el tejido del estroma. Además tinción puede llevarse a cabo para determinar si tales estructuras son verdaderamente prostática y de origen humano. 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

Los avances en la investigación del cáncer de próstata es muy limitada por la disponibilidad de sistemas de modelos de origen humano y de la hormona-naïve, que limitan nuestra capacidad de entender los eventos genéticos y moleculares que subyacen a la próstata inicio de la enfermedad. Hacia el desarrollo de mejores sistemas modelo para el estudio de la carcinogénesis de próstata humano, nosotros y otros han aprovechado la única potencial inductivo pro-prostática de embriones de roedores estroma prostático, denominada seno urogenital mesénquima (UGSM). Cuando se recombina con ciertas poblaciones de células pluripotentes tales como células madre embrionarias, UGSM induce la formación de epitelio humano normal de la próstata de una manera dependiente de la testosterona. Este sistema modelo humano puede utilizarse para investigar y probar experimentalmente la capacidad de los candidatos genes de susceptibilidad al cáncer de próstata a un ritmo acelerado en comparación con los estudios típicos de roedores transgénicos. Dado que las células madre embrionarias humanas (hESCs) pueden ser modificadas genéticamente en la cultura using de expresión génica inducible o siRNA vectores knock-down antes de la recombinación tejido, tal modelo facilita la prueba de las consecuencias funcionales de los genes, o combinaciones de genes, que se piensan para promover o impedir la carcinogénesis. La técnica de aislar poblaciones puras de células UGSM, sin embargo, es un reto y el aprendizaje a menudo requiere alguien con experiencia anterior para enseñar personalmente. Por otra parte, la inoculación de mezclas de células bajo la cápsula renal de un huésped inmunocomprometido puede ser técnicamente difícil. Aquí describimos e ilustramos el aislamiento adecuado de UGSM a partir de embriones de roedores y renal implantación cápsula de mezclas de tejidos para formar el epitelio prostático humano. Este enfoque, en su fase actual, requiere xenoinjerto in vivo de las células madre embrionarias; aplicaciones futuras podrían incluir potencialmente en la formación de la glándula o el uso in vitro de poblaciones de células madre pluripotentes inducidas (iPS).

Basándose en el informe apasionante por Taylor, et al., Nuestro laboratorio ha desarrollado un protocolo de injerto mediante el H1 de uso común (NIH-WA01 designado, genéticamente masculino) línea de células madre embrionarias humanas. 1 Esta línea ha sido rigurosamente probado para el control de calidad y es cariotipo normal de 13 Cuando se cultiva adecuadamente, HES células se pueden mantener, ampliar y criopreservados en un estado indiferenciado y pluripotente mediante un método ...

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

Para desentrañar los mecanismos moleculares que subyacen a la próstata primeros sistemas innovadores modelos humanos y enfoques cáncer de iniciación, la novela y se necesitan desesperadamente. El potencial de pre-prostática seno urogenital mesénquima (UGSM) para inducir a las poblaciones de células madre pluripotentes para formar el epitelio de próstata humano es una poderosa herramienta experimental en la investigación de próstata.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

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 vistas.  University of Chicago. Para desentrañar los mecanismos moleculares que subyacen a la próstata primeros sistemas innovadores modelos humanos y enfoques cáncer de iniciación, la novela y se necesitan desesperadamente. El potencial de pre-prostática seno urogenital mesénquima (UGSM) para inducir a las poblaciones de células madre pluripotentes para formar el epitelio de próstata humano es una poderosa herramienta experimental en la investigación de próstata.

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.

Trasplante de células madre en un In vitro Simulación de isquemia / reperfusión modelo

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

Investigación

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.

Inyección subretiniana de vectores de terapia génica y células madre en el ojo del ratón

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.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

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 modelo murino de cáncer de próstata humano Metástasis ortotópico

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.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

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

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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.

Evaluación de las propiedades de células madre en células de carcinoma de ovario humano utilizando multi e individuales basados ​​en células Esferas ensayos

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-captura microdisección de Human Prostática epitelio de Análisis de ARN

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.

Preclínica modelo murino ortotópico de cáncer de próstata humano

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.

Generación de un ratón humanizado hígado uso de células madre hepáticas humanas

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