Este trabajo fue apoyado por una beca de investigación integral en el Instituto de Medicina Regenerativa de California (RC1-00104), un Servicio de Salud Pública Grant (HL085377) del NHLBI, y un regalo de la Fundación Pollin de HSB

El protocolo escrito a continuación se basa en los parámetros publicados reportados por los autores, con algunas modificaciones. El protocolo se lleva a cabo con la aprobación de la UCSF Cuidado de Animales Institucional y Uso (IACUC) y de células madre de Supervisión de Investigación (SCRO) Comités. I. Cultura de células madre embrionarias humanas (hESCs) <ol><li> HESCs crecer en la norma 6-y (3,5 cm de diámetro, pozos) placas. </li><li> Por lo menos 12 horas antes de pases células de fibroblastos de embriones de ratón de semillas (MEF, obtenido a partir CF1 día E12.5 ratones tiempo-acoplado, irradiados en 1000 Ci) células alimentadoras en placas recubiertas con 1% de gelatina (6,7). MEFs debe ser plateado en la confluencia 50-60%, o 4 x 10 5 células por 3,5 cm de diámetro (véase figura 1). </li><li> Las células de paso cuando las colonias han llegado a la confluencia ~ 70% (Figura 2). </li><li> Al paso de las células, se aspira KSR medio de crecimiento (6) de los pozos y reemplazar con el medio de 0,5 ml que contiene colagenasa IV en KSR a una concentración de 1 mg / ml. </li><li> Incubar las placas con colagenasa IV a 37 ° C / 5% CO 2 durante 5 minutos o hasta que los bordes de las colonias empiezan a levantar de la placa. </li><li> Quitar colagenasa IV de la placa, añadir 0,5 ml de KSR a cada pocillo, y raspar las células de forma manual para eliminar por completo de la placa. </li><li> Recoger la suspensión de células de cada lugar, así, en un tubo cónico de 15 ml, y permiten que las células de resolver por la gravedad durante 5 minutos. </li><li> Eliminar el sobrenadante del tubo, dejando a las células y medianas empresas en un volumen total de ~ 300μl. </li><li> El uso de un 200 l micro-pipeta conjunto a todo volumen, suavidad pipeta de la suspensión de células 5-6 veces para romper las colonias. </li><li> Traiga la suspensión celular a un volumen total de 9 ml por pozo original de las células utilizando un medio de KSR (es decir, si un bien se cosecha, el tubo debe contener 9 ml). Complementar el medio con recombinante humano factor básico de crecimiento de fibroblastos (bFGF) a una concentración de 12,5 ng / ml. </li><li> Después de lavar los nuevos pozos que contienen células MEF (ver paso 2) con fosfato de Dulbecco buffer salino (DPBS) para eliminar todo el suero, añadir 3 ml de la suspensión celular a cada pocillo e incubar las placas a 37 ° C / 5% CO 2. </li><li> Cambio de medio KSR con bFGF 24 horas después de su paso, y cada 24-48 horas hasta que las células están listas para pasarse otra vez. </li></ol> II. Preparación de células madre de pellets para injertar <ol><li> A las 24 horas antes del injerto, agregar inhibidor de rock en el medio de KSR a una concentración final de 10μM para aumentar la viabilidad celular (8). Un pocillo de células en la confluencia ~ 70% (5 x 10 5 células) creará dos pastillas, con dos bolitas que se adhieren por cápsula renal. </li><li> Para preparar gránulos de las células para el injerto (9), se incuba la ROCA tratados con inhibidores de la colagenasa IV culturas durante 5 minutos a 37 ° C / 5% CO 2. </li><li> Aspirar la colagenasa IV del pozo y reemplazarlo con un DPBS ml con 10% de penicilina / estreptomicina (penicilina / estreptomicina). </li><li> Raspar manualmente las colonias de la placa, enjuague el bien con un adicional de 1 ml DPBS con penicilina / estreptomicina, y la transferencia de cada alícuota de 1 ml de suspensión celular a un tubo de microcentrífuga de 1,5 ml. </li><li> Girar los tubos de microcentrífuga brevemente a 8.000 x g, aspirar el sobrenadante y se resuspenden las pastillas en DPBS 500μl con penicilina / estreptomicina. </li><li> Para ayudar a unir las células en conjunto para el trasplante, añadir 100μl de 1 mg / ml de lectina Phaseolus vulgaris (PHA) en DPBS a cada tubo. </li><li> Se incuban las células / suspensión PHA a temperatura ambiente durante 5 minutos, luego girar durante 2 minutos a 8000 x g. Gire los tubos de 180 º dentro de sus pozos de rotor y girar de nuevo durante 2 minutos a 8000 x g. </li><li> Con cuidado, aspirar el sobrenadante dejando el sedimento intacto. </li><li> Desalojar a los gránulos de las células de los tubos pipeteando suavemente KSR 200μl junto al borde de la pastilla hasta que lentamente se levanta. </li><li> Una vez desalojados, transferir inmediatamente los pellets de 0.4μm inserta Millicell colocados en los pozos de 3,5 cm de diámetro que contiene 3 ml KSR, y se incuba durante 2 horas a la noche a 37 ° C / 5% CO 2. </li></ol> III. Cápsula renal injerto <ol><li> Realizar todos los procedimientos con animales, de acuerdo con las directrices institucionales utilizando una técnica aséptica apropiada. </li><li> Anestesiar a los ratones SCID beige (CB17.Cg-Prkdc scid Lyst bg / Crl), con edades entre 8-12 semanas, utilizando una combinación de O2 inhalado 3% isoflurane/0.5% y tribromoetanol intraperitoneal (Avertin, 25 mg / kg recién preparada). Dos gránulos de las células por ratón será implantada utilizando las técnicas descritas (10). </li><li> Después de colocar el ratón sobre una almohadilla de calefacción para prevenir la hipotermia, afeitar el área de la incisión, y luego desinfectar con clorhexidina. Haga una incisión de 2,5 cm a través de la piel junto al centro, pero en paralelo a la columna vertebral. </li><li> Mantener la humedad del área de la incisión con agua estéril saline, hacer una pequeña incisión a través del músculo, justo debajo de las costillas y ~ 1 cm de la columna vertebral. La incisión debe ser lo suficientemente grande como para sacar el riñón a través de, pero lo suficientemente pequeño que el riñón no se deslice hacia la cavidad abdominal sin fuerza. </li><li> Tire suavemente el riñón a través de la incisión de los músculos con una pipeta Pasteur de vidrio que ha sido puesto en la forma de un gancho romo, ligeramente curvada. </li><li> Mojar el riñón con DPBS estéril para evitar la cápsula se seque. Esto puede ser necesario repetir todo el procedimiento, como la cápsula es muy frágil. </li><li> Utilizando unas pinzas Dumont n º 5, tire suavemente de la cápsula desde el riñón y hacer una incisión de 2 mm con unas tijeras Vannas primavera. </li><li> Con otra pipeta de vidrio que ha sido puesto en una muy fina, la sonda roma, hacer un pequeño hueco entre la cápsula y el riñón. </li><li> Usando una pipeta de gran orificio colocado en una pipeta de transferencia disponibles, tomar una sola célula de pellets a partir de la inserción Millicell. Asegúrese de tomar tan poco los medios de comunicación adicional a lo largo como sea posible, ya que el exceso de material puede interferir con la transferencia de pellets. </li><li> Mientras se mantiene hasta el borde de la incisión para crear el bolsillo, coloque suavemente el pellet al lado de la apertura y empuje en el bolsillo hacia un polo del riñón con la pipeta tiró. </li><li> Repita el proceso con una segunda pastilla. Lugar esta bajo la cápsula del riñón mismo, pero hacia el polo opuesto. </li><li> Con cuidado, coloque la cápsula de vuelta a las pastillas. Los gránulos deben permanecer dentro de la bolsa, y no las suturas son necesarias para cerrar la cápsula. </li><li> Empuje suavemente el riñón en la cavidad abdominal y cierre de la pared muscular con suturas de seda 4-0 (o menor) con adjuntos inversa corte aguja. La incisión debe ser lo suficientemente pequeño que sólo una única sutura es necesario. </li><li> Cerca de la piel con una serie de 4-5 puntos sueltos, utilizando el mismo material de sutura. </li><li> Antes de colocar el ratón de nuevo en su jaula, administrar 0,05 mg / kg de buprenorfina (11,12) y 5 mg / kg de ketoprofeno (12,13) ​​como analgésico a corto plazo y largo plazo, respectivamente. Sustancias controladas sólo se utiliza de acuerdo con las directrices institucionales. </li><li> La transferencia de los animales de vuelta a su jaula y se permite que se recupere de la anestesia en una manta eléctrica. Esto debe tomar alrededor de 20-30 minutos. </li><li> Una vez que el ratón es totalmente ambulatoria, que pueden ser devueltos a las instalaciones de alojamiento de los animales. </li></ol> IV. Cosecha teratoma, la fijación y seccionamiento <ol><li> En 8-12 semanas después de la cirugía, debe haber un teratoma apenas palpable cerca del riñón. Grandes, quistes llenos de líquido a menudo se desarrollan, así, a partir de las 6-8 semanas. Estos pueden requerir una cosecha más temprana si causan trastornos a los animales. Desde teratomas creciendo a un ritmo más lento que los quistes, sin embargo, tendrá que esperar el mayor tiempo posible para obtener un teratoma de tamaño suficiente para el análisis (es decir, por lo menos 8 semanas). </li><li> Extirpar el riñón, el teratoma, y ​​cualquier quiste rodea como un bloque de tejido de la cavidad abdominal utilizando el mismo método quirúrgico mediante el cual se accede al riñón original (véase la sección III, los pasos 1-4 arriba). </li><li> Coloque el bloque de tejido extraído en un tubo que contiene 10% de formol. </li><li> Deje que el teratoma para arreglar la noche a 4 ° C. </li><li> Al día siguiente, retire el teratoma del tubo. Diseccionar los quistes y los riñones tanto como sea posible sin dañar el teratoma en sí mismo (Figura 3). Mayor teratomas pueden ser cortados por la mitad para facilitar la disección y la inclusión. </li><li> Proceso del teratoma utilizando un estándar de parafina fijación técnica. Unidades automatizadas están disponibles (por ejemplo, hipercentro Shandon): <table border="0"><tbody><tr><td> I. </td><td> 80% de Flex alcohol </td><td> 2 horas </td></tr><tr><td> II. </td><td> 80% de Flex alcohol </td><td> 1 hora </td></tr><tr><td> III. </td><td> 85% de Flex alcohol </td><td> 1,5 horas </td></tr><tr><td> IV. </td><td> 95% de Flex alcohol </td><td> 1 hora </td></tr><tr><td> V. </td><td> Flex 100% alcohol </td><td> 1 hora </td></tr><tr><td> VI. </td><td> Flex 100% alcohol </td><td> 1 hora </td></tr><tr><td> VII. </td><td> Flex 100% alcohol </td><td> 1 hora </td></tr><tr><td> VIII. </td><td> Claro-Rite 3 </td><td> 1 hora </td></tr><tr><td> IX. </td><td> Claro-Rite 3 </td><td> 1 hora </td></tr><tr><td> X. </td><td> Claro-Rite 3 </td><td> 1 hora </td&gt; </tr><tr><td> XI. </td><td> Parafina (TissuePrep) </td><td> 2 horas </td></tr><tr><td> XII. </td><td> Parafina (TissuePrep) </td><td> 2 horas </td></tr></tbody></table></li></ol> V. La inmunohistoquímica y análisis <ol><li> Una vez incluidos en parafina, la sección de teratoma en 5μm cortes seriados con un micrótomo rotatorio, y las secciones de flotar en las diapositivas. </li><li> Antes de la tinción, los portaobjetos para cocer al menos una hora. </li><li> Tinción de las láminas con hematoxilina y eosina con los métodos estándar para identificar las estructuras de tejido representante de cada una de las tres capas germinales embrionarias (Figura 4): <table border="0"><tbody><tr><td> I. </td><td> Lavado de xileno </td><td> 3-5 minutos </td></tr><tr><td> II. </td><td> Xileno </td><td> 3-5 minutos </td></tr><tr><td> III. </td><td> 100% de alcohol </td><td> 3-5 minutos </td></tr><tr><td> IV. </td><td> 100% de alcohol </td><td> 3-5 minutos </td></tr><tr><td> V. </td><td> 95% de alcohol </td><td> 3-5 minutos </td></tr><tr><td> VI. </td><td> 80% de alcohol </td><td> 3-5 minutos </td></tr><tr><td> VII. </td><td> Agua de lavado (varios cambios) </td><td> 2 minutos </td></tr><tr><td> VIII. </td><td> Hematoxilina de Gill # 3 </td><td> 2-5 minutos </td></tr><tr><td> IX. </td><td> Agua de lavado (varios cambios) </td><td> hasta que el agua salga clara </td></tr><tr><td> X. </td><td> Agua de Scott (a los núcleos azules) </td><td> 3 minutos o más </td></tr><tr><td> XI. </td><td> Agua de lavado (varios cambios) </td><td> 1-2 minutos </td></tr><tr><td> XII. </td><td> Eosina </td><td> 1 minuto </td></tr><tr><td> XIII. </td><td> Agua de lavado (varios cambios) </td><td> 30 segundos </td></tr><tr><td> XIV. </td><td> 80% de alcohol </td><td> 1 minuto </td></tr><tr><td> XV. </td><td> 95% de alcohol </td><td> 2 minutos </td></tr><tr><td> XVI. </td><td> 95% de alcohol </td><td> 2 minutos </td></tr><tr><td> XVII. </td><td> 100% de alcohol </td><td> 3 minutos </td></tr><tr><td> XVIII. </td><td> 100% de alcohol (limpia) </td><td> 3 minutos </td></tr><tr><td> XIX. </td><td> Xileno </td><td> 2 minutos o más </td></tr><tr><td> XX. </td><td> Xileno </td><td> 2 minutos o más </td></tr></tbody></table></li><li> Montar un cubreobjetos sobre cada diapositiva con Permount. </li></ol> VI. Resultados representante <img src="/files/ftp_upload/2036/2036fig1.jpg" alt="Figura 1" /> Figura 1. Revestimiento de elementos combustibles irradiados CF1 fibroblastos embrionarios de ratón como una capa de conexión para el cultivo de hESCs indiferenciada. MEFs se colocan en la confluencia de ~ 50%, como se muestra aquí, o 4 x 10 5 células por 3,5 cm de diámetro y en una placa de cultivo de 6 pocillos. Bar, 100 micras. <img src="/files/ftp_upload/2036/2036fig2.jpg" alt="Figura 2" /> Figura 2. HESCs ejemplo de la cultura subconfluentes de hESCs indiferenciadas en una capa de alimentación MEF. Se cultivan en las capas de alimentación MEF hasta ~ 70% de confluencia, como se muestra aquí, como se describe pasajes. Bar, 100 micras. <img src="/files/ftp_upload/2036/2036fig3.jpg" alt="Figura 3" /> Figura 3. Ejemplo de teratomas explantados. Después de la explantación, el teratoma, el riñón y los tejidos accesorio se fija como un bloque en formol, el riñón y el tejido de accesorios son cuidadosamente recortada antes de inclusión en parafina. <img src="/files/ftp_upload/2036/2036fig4.jpg" alt="Figura 4" /> Figura 4. Los teratomas derivados de hESCs indiferenciada contienen representante de los tejidos de todas las tres capas germinales en vivo. Un teratoma, como una muestra en la Figura 3, se seccionó y se tiñó con hematoxilina y eosina para identificar los tejidos embrionarios. hESCs dar lugar a tejidos derivados de las tres capas germinales embrionarias (ectodermo (A, B), el mesodermo (C), endodermo (D). (A) la estructura naciente del tubo neural. (B) epitelio escamoso primitivo. (C) El cartílago rodeado por una cápsula de mesénquima condensado. (D) la estructura glandular intestinal. Bar, 100 micras. Imágenes reimpreso con el permiso del autor. <img src="/files/ftp_upload/2036/2036fig5.jpg" alt="Figura 5" /> Figura 5. Análisis de teratoma puede ser utilizado para trazar el destino de los etiquetados, hESCs indiferenciada. Como ya se ha publicado (9), una mezcla de hESCs específicamente etiquetados con hESCs sin etiqueta se puede utilizar para trazar el destino de las células marcadas en un ensayo de formación de teratoma. Como se muestra aquí, hESCs indiferenciadas que expresan el marcador de superficie, CD133, y etiquetada con el aumento de proteína verde fluorescente (EGFP), se mezclaron con hESCs sin etiquetar, indiferenciado. Estos fueron utilizados para formar teratomas por la cápsula renal del injerto. El análisis inmunohistoquímico de hematoxilina y eosina secciones teñidas con teratoma con anti-eGFP anticuerpos demuestra el destino neuroectodérmico de la CD133 + hESCs. Los teratomas fueron procesados ​​como en la Figura 4 y contrastados con anticuerpos anti-eGFP (marrón) para localizar los derivados de células CD133 + GFP + dentro de los tejidos. CD133 + derivadas de las células (flechas) se observaron en los tejidos derivados del ectodermo embrionario, el epitelio neural específica (izquierda) y de la naciente neural, como estructuras tubulares (derecha). Bar, 100 micras. Imágenes reimpreso con el permiso del autor. Gelatina <ul><li> 0,1% de gelatina porcina </li><li> DPBS 99,9% <ul><li> Agregue la gelatina a DPBS, se incuba a 37 ° C durante una hora o hasta que la gelatina se disuelve, y esterilizar filtro (0.22μm) antes de su uso. </li></ul></li></ul> MEF medio <ul><li> 10% de suero fetal bovino (calificados) </li><li> 90% de Dulbecco modificado del medio esenciales (D-MEM) </li><li> 1x L-Glutamina </li><li> 1x penicilina / estreptomicina <ul><li> Filtro de esterilización antes de su uso. </li></ul></li></ul> KSR (sustitución de golpe de gracia en suero) medio <ul><li> 20% de sustitución de suero golpe de gracia </li><li> 80% golpe de gracia D-MEM </li><li> 1x L-Glutamina </li><li> 1x aminoácidos no esenciales </li><li> 0,1 mM β-mercaptoetanol / </li><li> 12.5 ng / ml de bFGF <ul><li> Filtro de esterilización. </li><li> Almacenar a 4 ° C, pero cálido a 37 ° C antes de añadir a las células. </li><li> Añadir bFGF inmediatamente antes del uso. </li></ul></li></ul> Colagenasa IV <ul><li> Disolver 1 mg / ml en medio de KSR, colocando a 37 ° C durante 1-2 horas o hasta que todo el polvo se disuelve. </li><li> Filtro de esterilización. </li></ul> Avertin <ul><li> 0,25 g de 2,2,2-tribromoetanol </li><li> 0,25 ml de 2-metil-2-butanol <ul><li> Se incuba a 37 ° C durante la noche para formar 100% de la solución. </li><li> Varias horas antes de la cirugía, solución diluida al 2,5% de trabajo con DPBS y se incuba a 37 ° C durante al menos 1 hora. </li><li> Filtro de esterilización y alícuota. </li><li> Tribromoetanol es altamente sensible a la luz, por lo que protegerlo de la luz en todas las etapas de preparación y uso. </li><li> Preparar no más de 12-24 horas antes de su uso. </li></ul></li></ul> La buprenorfina <ul><li> 1 mg / ml de solución stock en metanol <ul><li> Diluir a 0,015 mg / ml de solución estéril de trabajo utilizando H 2 O. </li><li> Almacenar a -20 ° C hasta por 1 mes. </li></ul></li></ul> Ketoprofeno <ul><li> 0,5 mg / ml de solución de trabajo en DPBS <ul><li> Añadir DPBS, incubar durante la noche a 37 ° C durante 1-2 horas o hasta que todo el polvo se disuelve. </li><li> Filtro de esterilizar y almacenar a 4 ° C. </li></ul></li></ul>

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C., Ducrot, R., Pasquet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+pharmacological+and+toxicological+studies+on+ketoprofen.">Some pharmacological and toxicological studies on ketoprofen.</a> Rheumatol Rehabil. , (1976).</li></ol>

Hemos adaptado un ensayo de formación de teratomas altamente eficiente para la elaboración de mapas de la suerte de diferenciar hESCs en un entorno en vivo. Un pequeño número de hESCs específicamente seleccionados se mezcla con indiferenciado hESCs de tipo salvaje y lectina de Phaseolus vulgaris para formar un pellet de células. Esto es injertado debajo de la cápsula renal en un ratón inmunodeficiente. El destino de la hESCs seleccionado originalmente, se puede determinar mediante técnicas de inmunohistoquímica (Figura 5), como se informó anteriormente (9). Este método proporciona una valiosa herramienta para la identificación y la generación de tejidos reactivos específicos para la terapia basada en células.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2-Methyl-2-butanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>240486</td>
<td>Avertin</td>
</tr>
<tr>
<td>2,2,2-Tribromoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T48402</td>
<td>Avertin</td>
</tr>
<tr>
<td>4-0 silk sutures</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>641G</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>233FB</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B7536</td>
<td>&nbsp;</td>
<tr>
<td>Clear-Rite 3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046-341</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>&nbsp;</td>
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>Eosin Y</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-245-658</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (Qualified)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Flex alcohol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046344</td>
<td>&nbsp;</td>
<tr>
<td>Gill&#x2019;s Hematoxylin #3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-050-204</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
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<tr>
<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
</tr>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K2012</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25020-081</td>
<td>&nbsp;</td>
<tr>
<td>MILLICELL inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM01250</td>
<td>&nbsp;</td>
<tr>
<td>Nonessential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
</tr>
<tr>
<td>Paraffin (TissuePrep)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>8002-74-02</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L1668</td>
<td>&nbsp;</td>
<tr>
<td>Porcine gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G6144</td>
<td>&nbsp;</td>
<tr>
<td>ROCK inhibitor</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>Y-27632</td>
<td>&nbsp;</td>
<tr>
<td>SCID beige mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>250</td>
<td>&nbsp;</td>
<tr>
<td>Scott&#x2019;s Wash</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>6697-32</td>
<td>Ricca Chemicals</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fate mapping of human embryonic stem cells by teratoma formation

Células madre embrionarias humanas (hESCs) tienen una capacidad ilimitada para la auto-renovación, y la capacidad de diferenciarse en células derivadas de las tres capas germinales embrionarias (1). Diferenciación dirigida de hESCs en tipos celulares específicos ha generado mucho interés en el campo de la medicina regenerativa (por ejemplo, (2-5)), y los métodos para determinar la<em&gt; In vivo</emDestino&gt; de hESCs seleccionar ni manipular son esenciales para este esfuerzo. Hemos adaptado un ensayo de formación de teratomas altamente eficiente para este propósito. Un pequeño número de hESCs específicamente seleccionados se mezcla con indiferenciado hESCs de tipo salvaje y lectina de Phaseolus vulgaris para formar un pellet de células. Esto es injertado debajo de la cápsula renal en un ratón inmunodeficiente. Tan sólo 2,5 x 10<sup&gt; 5</supHESCs&gt; se necesitan para formar una de 16 cm<sup&gt; 3</sup&gt; Teratoma en 8-12 semanas. El destino de la hESCs seleccionado originalmente, se puede determinar mediante técnicas de inmunohistoquímica. Este método proporciona una valiosa herramienta para la caracterización de los tejidos reactivos específicos para la terapia basada en células.

Watch this Scientific Journal Video about Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation at JoVE.com

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Diferenciación dirigida de células embrionarias humanas en específico ha generado mucho interés en la medicina regenerativa. Ofrecemos una concisa, paso a paso el protocolo para determinar la<em&gt; In vivo</em&gt; Suerte de hESCs seleccionado que proporciona una valiosa herramienta para la caracterización de los tejidos reactivos específicos para la terapia basada en células.

El destino de la asignación de células estaminales embrionarias humanas mediante la formación de teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

fate-mapping-of-human-embryonic-stem-cells-by-teratoma-formation

Research

JoVE Journal

Biology

17.1K vistas.  University of California San Francisco. Diferenciación dirigida de células embrionarias humanas en específico ha generado mucho interés en la medicina regenerativa. Ofrecemos una concisa, paso a paso el protocolo para determinar la In vivo Suerte de hESCs seleccionado que proporciona una valiosa herramienta para la caracterización de los tejidos reactivos específicos para la terapia basada en células.

Video: Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

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

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

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

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Sondeo de la actividad mitocondrial complejo en células estaminales embrionarias humanas

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

En el etiquetado in vitro de células estaminales embrionarias humanas de Resonancia Magnética

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparación Extienda los cromosomas de las células estaminales embrionarias para cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...