Nos gustaría reconocer el amplio FACS de células madre del Centro de Investigación y sobre todo gracias a Jessica Scholes y Codrea Felicia por su ayuda con la clasificación de células. El trabajo fue financiado por el CIRM RN2-00904-1, K08 HL074229, American Thoracic Society / COPD Foundation ATS-06-065, La Fundación preocupación, la UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology Program / SPORE pulmón Cáncer, la Universidad de California del Cáncer Investigación Comité de Coordinación y el Gwynne Hazen cereza Memorial Laboratories (BG).

Esquema de los pasos 1. La disección de la tráquea 2. Limpieza de la tráquea y se corta 3. Enzima digestión y el tratamiento en suspensión de células individuales 4. La tinción de FACS y clasificación 5. Procesamiento de células clasificadas para in vivo e in vitro modelos 1. La disección de la tráquea <ol><li> Sacrificar al ratón con una inyección intraperitoneal de 0,1 cc mg/0.2 de pentobarbital. </li><li> Cortar abrir la pared abdominal, y mover los intestinos al lateral para exponer la aorta abdominal y luego disecado. </li><li> Abrir el pecho por el corte a través del diafragma y de la pared torácica anterior en ambos lados para exponer el corazón y los pulmones. Continuar el corte en el cuello de la eliminación de las glándulas salivales y la grasa para exponer la tráquea. </li><li> Retire el timo y cortar el pulmón de sus conexiones por debajo y por detrás. Separcomió la tráquea del esófago mediante la inserción de una pinza entre ellos luego se corta la tráquea en su parte más superior, por encima de su conexión con la laringe. A continuación, mantenga la tráquea con fórceps y disecado de su unión y continuar hacia abajo para quitarla con el corazón y los pulmones en bloque. Recorte el corazón y los pulmones cerca del hilio incluir la mayoría de los bronquios principales derecho e izquierdo. </li><li> Coloque la tráquea en una pequeña placa Petri que contenía medio de recogida en hielo. </li></ol> 2. Limpieza de la tráquea y se corta <ol><li> Bajo el microscopio de disección, utilice pinzas punta muy fina (N º 5) y tijeras Vannas Tubingen (FST 15003-08 finescience.com) para limpiar la tráquea de todos los tejidos que están conectados a la misma. Esto incluye: los ganglios linfáticos, el nervio laríngeo recurrente, la grasa, la glándula tiroides y el resto de los vasos pulmonares. La parte superior de la tráquea necesita una atención especial, para eliminar los cartílagos laríngeos grandes sin comprometer el mayor grupo de SMGque se presente entre el cartílago cricoides y el primer anillo traqueal cartilaginosos (C1) 4. </li><li> Cortar la tráquea en la parte superior y la parte inferior entre C4 y C5 y luego se corta abierta ambas partes a través de la luz de la tráquea. </li></ol> 3. Enzima Digestión y Transformación en suspensión de células individuales (SCS) <ol><li> Incubar la parte inferior de la tráquea en 1 ml de 16 U de dispasa en un tubo Eppendorf a temperatura ambiente (RT) durante 30 min, como se ha descrito previamente 5. A continuación, añadir 0,5 mg / ml de DNasa I en DMEM durante 20 min a TA. </li><li> Eliminar las tráqueas digeridos en medio fresco recogida en una placa de Petri estéril, y pelar el epitelio de la superficie bajo el microscopio de disección. Utilizar una pipeta para transferir el medio que contiene el epitelio despojado a un tubo cónico de 50 ml. </li><li> Decantar el epitelio separa por arrastre a 1.000 xg, a 4 ° C. Eliminar el sobrenadante y añadir 0,1% de tripsina / EDTA y se incuba con agitación the en un tubo de 37 ° C incubadora con agitación (200 rpm) durante 30 min. Después de la incubación, usando una punta de 1.000 l, pipetear arriba y hacia abajo para romper los grumos y formar una suspensión de células individuales. </li><li> Coloque la tráquea superior en un tubo cónico que contenía 1 ml de 0,15% pronasa. Luego incubar a 4 ° C durante 4 horas. </li><li> Vórtice suavemente el tubo y luego colocar la tráquea en medio de recogida fresco y confirmar bajo el microscopio de disección que hay desprendimiento completo de la superficie del epitelio (SE). </li><li> Picar la tráquea con unas tijeras pequeñas para abrir los compartimientos SMG y luego incubar durante una hora más en pronasa a TA con agitación lenta. </li><li> Decantar el tejido traqueal, eliminar la pronasa, añadir 0,1% de tripsina / EDTA y se incuba a 37 ° C durante 30 min con agitación como anteriormente. </li><li> Pasaje de las células digeridas, macizos y cualquier trozos restantes de las glándulas a través de agujas 20G, 23G y 26G utilizando una jeringa de 10 ml. Repita las veces varios pases con cada tamaño de la aguja antes decese. Ahora, la mayoría de las células deben estar en un SCS. </li><li> Se filtra la suspensión a través de un filtro de 40 mM y luego lavar el filtro y centrifugar las células. A continuación, reconstituir las células en el volumen adecuado de medio, contar las células y proceder inmediatamente a la tinción FACS y clasificación. No deje las células en 4 ° C o en hielo durante varias horas porque encontramos que esto afecta a la viabilidad y reduce gravemente la esfera que forma la eficiencia. </li></ol> 4. La tinción de FACS y clasificación <ol><li> Tome suficientes células para la preparación de los tubos sin teñir y teñidas sola mancha de compensación entonces el resto de las células con anticuerpos primarios, Trop2 de cabra y de rata ITGA6, durante 15 min a RT. Entonces lavar con PBS o medio de recogida, removedor el sobrenadante cuidadosamente y romper el sedimento celular. </li><li> Tiñen con el anticuerpo secundario adecuado, incubar en la oscuridad a temperatura ambiente durante 10 min, lavar y eliminar el sobrenadante y luego romper la pastilla. </li><li> En el FACSARIA, después de ajustar el voltaje y la indemnización, puerta de los escombros, las células muy pequeñas, dobletes celulares y células muertas. Entonces, la mayoría de las células de la SE debe ser TROP2 positivo mientras que 20-50% de las células SMG será TROP2 + 6. </li><li> Establecer la puerta de clasificación para Trop2 + ITGA6 + células de SE (células basales) y para Trop2 + células (+ /-ITGA6 células positivas) de SMG (células de los conductos). </li></ol> 5. Procesamiento de Células clasificadas: en cultivo in vitro y el modelo in vivo 5.1 En el ámbito de la formación in vitro 5-9 cultura <ol><li> Mezclar 100 l de &quot;factor de crecimiento reducido Matrigel&quot; con 100 l de medio completo 10,11 (ver Tabla 1) que contiene hasta 50.000 células clasificadas a continuación, poner la mezcla en la cámara superior de un 24-así trans-bien. A continuación, poner 400 l de medio completo en la cámara baja y se incuba a 37 ° C. Cambio medio en la cámara baja cada dos días y añadir otros 100 l de Matrigelcada semana o antes si se adelgaza. </li></ol> 5.2 En el modelo in vivo <ol><li> Reconstituir la ordenados pellet de células en medio completo mezclado 1:1 con Matrigel a una concentración de 5,000-10,000 células por 50 l. </li><li> Anestesie los receptores C57/BL6 ratones. Administrar Carprofen (5 mg / kg) subcutaenously como analgésico. Esterilice y afeitarse la espalda luego hacer una incisión longitudinal en el centro de la piel de la espalda superior. La incisión debe ser lo suficientemente grande como para identificar el lugar de la almohadilla de inyección de grasa, así como la ampolla que se forma después de la inyección. Utilice el lado romo de la tijera para ensanchar las bolsas entre la piel y el reverso de la pared torácica en ambos lados para maximizar el acceso y la visualización. </li><li> Identifique el borde medial de la escápula, empujando la extremidad anterior del ratón hacia arriba y hacia atrás para ver el movimiento escápula. Tomar 50 l de la suspensión celular en un 300 l jeringa de insulina BD y lo inyecta en la almohadilla grasa medial a la lateral de la escápula ylas vértebras. Usted debe ser capaz de ver una ampolla de lo contrario son demasiado profundas. Repita lo mismo en el otro lado luego cortar o coser la incisión de la piel. Entrada ratones y dar Carprofen (5 mg / kg, SC) una vez al día durante 48 hr. </li><li> Después de 3 semanas, la eutanasia a los ratones con pentobarbatol (0,1 mg/0.2 cc, IP), abra la piel como antes y diseccionar la almohadilla de grasa total por ambos lados con un margen de seguridad para evitar la pérdida de cualquier estructura de tejido que podría haberse formado y se extendió alrededor. Fijar en PFA y de inserción en parafina 12. El cuidado extremo debe ser empleado durante el recorte y el corte del bloque de no omitir o pasar por alto una estructura. La mejor práctica consiste en recortar 40 micras una vez y luego cortar una rebanada de 4 micras, lo coloca en un portaobjetos de vidrio y examinarlo bajo el microscopio para confirmar si hay &quot;estructuras epiteliales&quot; en esta parte del bloque o no. Si no hay nada visto, entonces recortar una vez más y repita este paso. </li></ol> 6. Los resultados representativos Stripping de las tráqueas de ratón se traducirá en una tráquea denudado que es visible por microscopía de luz como se muestra en la Figura 1A. Figura 1B muestra la vista de campo brillante de una tráquea después epitelial stripping con glándulas submucosas de ser liberado de los tejidos y los racimos de uvas parecidas. Pronasa elimina el epítopo ITGA6 y esta es la razón por la TROP2 células de los conductos no se pueden separar en ITGA6 + y - poblaciones. Sin embargo, dispasa, que conserva el epítopo ITGA6, digiere todo el epitelio de la superficie y las células de los conductos, incluso con tiempos de incubación muy corto, y por lo tanto no se puede utilizar para separar las células basales de conducto 3. La citometría de flujo de las suspensiones de células individuales deben mostrar gráficos de dispersión hacia delante y dispersión lateral que se representan en la Figura 2A. Buena separación de células de los conductos del resto si las células traqueal debe ser visto con el anticuerpo Trop2 en citometría de flujo para células activadas por fluorescencia sorting (FACS) como se ve en la Figura 2B. Esferas debe ser visible en el matrigel después de aproximadamente 1 semana en cultivo y son densos en apariencia, como se muestra en la Figura 3. La eficiencia de este proceso es del orden de 1-2% y las células individuales estará aún presente en el matrigel y probablemente representan células de los conductos de las glándulas submucosas que no poseen la capacidad de auto-renovación. La inyección de células madre del conducto en los resultados cojín de ratón de grasa en la formación de la glándula submucosa, como estructuras que se muestran la figura 4. Estos son vistos en los bloques, incluso sin tinción H &amp; E. Estos son esféricas y poseen un lumen central. Muchas secciones transversales deben hacerse a través de la almohadilla de grasa con el fin de no perder estas estructuras epiteliales. <img alt="Figura 1" src="/files/ftp_upload/3731/3731fig1.jpg" /> Figura 1. La digestión enzimática del epitelio traqueal. La eliminación de las células epiteliales de la superficie se muestra con blacflechas k, la eliminación de las células en los conductos de las glándulas submucosas se muestra con las flechas verdes. Tráqueas denudados que son visibles al microscopio óptico se muestran después de 30 minutos de digestión dispasa y después de 4 horas de digestión con pronasa. <img alt="Figura 2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> La Figura 2A. I. Citometría de flujo de las suspensiones de células individuales que muestran dispersión representativo adelante y parcelas dispersión lateral de tráqueas de ratón después de la superficie epitelial de extracción. ii. Parcelas FACS representativos para TROP2 expresión en células de los conductos de tráqueas ratón. <img alt="Figura 2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> La Figura 2B. I. Citometría de flujo de las suspensiones de células individuales de la superficie del epitelio despojado de los dos tercios inferiores de la tráquea de ratón que muestra la dispersión hacia adelante y representante parcelas dispersión lateral. ii. FACS trama típica de ITGA6 expresión in surface epitelio. iii. Otra FACS gráfico representativo de adelante y dispersión lateral de la superficie del epitelio que muestra de nuevo gating de la población de células basales en verde. iv. Típica ITGA6 y TROP2 población de células que expresan en verde. <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig3.jpg" /> Figura 3. Transcurso de tiempo de desarrollo de las esferas. A. Conducto esferas. Esferas debe ser visible en el matrigel después de aproximadamente 1 semana en cultivo y casi todos son densos en apariencia. Las células individuales que no forman esferas también se ven. (Barra de escala = 50 mm). Esferas B. basales son más grandes y luminal en apariencia. Las células individuales que no forman esferas también se ven. (Barra de escala = 50 mm). <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig4.jpg" /> Figura 4. La inyección de células madre del conducto en los resultados cojín de ratón de grasa en la formación de la glándula submucosa como estructuras. Estructuras representativasse muestran en la almohadilla de grasa y algunos se muestran producir mucina (luz azul) con la Alcian azul ácido periódico de Schiff mancha.

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Esta técnica para aislar células de los conductos y basal de las vías respiratorias es importante para mejorar nuestra comprensión de la reparación de las vías respiratorias y las enfermedades de las vías respiratorias y regeneración. Las técnicas descritas aquí son algunas medidas críticas. El primero es el período optimizado digestión enzimática. La segunda es la creación de una única suspensión celular a través de pasada en serie con agujas de calibre progresivamente más altas para prevenir la rotu...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> El número de catálogo </td></tr><tr><td> Medio completo 10: DMEM-F-12, 50/50, 1X) </td><td> Mediatech </td><td> 15 a 090-CV </td></tr><tr><td> Hepes (15 mM) </td><td> Invitrogen </td><td> 15630 </td></tr><tr><td> Bicarbonato de sodio (3,6 mm o 0,03%) </td><td> Invitrogen </td><td> 25080 </td></tr><tr><td> L-glutamina (4 mM) </td><td> Mediatech </td><td> 25-005-Cl </td></tr><tr><td> Penicilina (100 U / ml) </td><td> Mediatech </td><td> 30 a 001-CI </td></tr><tr><td> Estreptomicina (100 mg / m) </td><td> Mediatech </td><td> 30 a 001-CI </td></tr><tr><td> La anfotericina B (0,25 g / ml) </td><td> Lonza </td><td> 17-836R </td></tr><tr><td> Eninsulina (10 mg / ml) </td><td> Sigma </td><td> I6634 </td></tr><tr><td> Transferrina (5 mg / ml) </td><td> Sigma </td><td> T1147 </td></tr><tr><td> La toxina del cólera (0,1 g / ml) </td><td> Sigma </td><td> C8052 </td></tr><tr><td> Factor de crecimiento epidérmico (25 ng / ml) </td><td> BD </td><td> 354001 </td></tr><tr><td> Extracto de pituitaria bovina (30 mg / ml) </td><td> Invitrogen </td><td> 13028-014 </td></tr><tr><td> Suero bovino fetal (5%) </td><td> Pescador </td><td> SH3008803HI </td></tr><tr><td> El ácido retinoico (0,05 mM) </td><td> Sigma </td><td> R2625 </td></tr><tr><td> Factor de crecimiento reducido Matrigel </td><td> BD </td><td> 354230 </td></tr></tbody></table> Tabla 1. Completar componentes de medios. <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td&gt; <td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Pronasa </td><td> Roche </td><td> 10165921001 </td><td> Utilizado en un 0,15%: -O / N a 4 ° C para aislar la digestión total de células de la tráquea (para cultivo ALI) -4 Digestión hr 4 ° C para aislar SMG </td></tr><tr><td> Dispasa </td><td> BD Biosciences </td><td> 354235 </td><td> Usado en 16 Unidades: 30 min a RT </td></tr><tr><td> DNasa I </td><td> Sigma </td><td> DN25 </td><td> Usado a 0,5 mg / ml: 20-30 min a RT </td></tr></tbody></table> Enzimas Tabla 2. Utilizado para la digestión enzimática de la tráquea.

isolation of basal cells and submucosal gland duct cells from mouse trachea

Las vías respiratorias están directamente en contacto con el medio ambiente y por lo tanto susceptibles a las lesiones de las toxinas y agentes infecciosos que se respira en 1. Las vías respiratorias grande por lo que requieren un mecanismo de reparación eficaz para proteger a nuestros cuerpos. Este proceso de reparación se produce a partir de células madre en las vías respiratorias y el aislamiento de estas células madre de las vías respiratorias es importante para la comprensión de los mecanismos de reparación y regeneración. También es importante para la comprensión de reparación anormal que puede conducir a enfermedades de las vías 2. El objetivo de este método consiste en aislar una población de células madre de ratón novela de los conductos de las glándulas submucosas traqueales y para colocar estas células in vitro y en modelos de sistemas in vivo para identificar los mecanismos de reparación y regeneración de las glándulas submucosas 3. Esta producción muestra métodos que pueden ser utilizados para aislar y ensayo de las células madre del conducto y basal de la gran vías respiratorias 3. Esto nos permitirápara estudiar las enfermedades de las vías respiratorias, tales como la fibrosis quística, el asma y la enfermedad pulmonar obstructiva crónica. En la actualidad, no existen métodos para el aislamiento de células de las glándulas submucosas y conductos no hay modelos in vivo para estudiar la regeneración de las glándulas submucosas.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Aquí demostramos nuestro protocolo de aislamiento de las células de los conductos glandulares basales y submucosa de tráqueas de ratón. También se demuestra el método de la inyección de células madre en la alfombrilla del ratón grasa dorsal para crear un<em&gt; In vivo</em&gt; Modelo de regeneración glándula submucosa.

Aislamiento de células basales y de células submucosas conducto de la glándula de ratón tráquea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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13.3K vistas.  David Geffen School of Medicine at UCLA. Aquí demostramos nuestro protocolo de aislamiento de las células de los conductos glandulares basales y submucosa de tráqueas de ratón. También se demuestra el método de la inyección de células madre en la alfombrilla del ratón grasa dorsal para crear un In vivo Modelo de regeneración glándula submucosa.

Video: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Aislamiento de células T CD4 + de los ganglios linfáticos del ratón utilizando Miltenyi Purificación MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Investigación

Biología

Isolation and Derivation of Mouse Embryonic Germinal Cells

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

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

El aislamiento y la derivación de células embrionarias de ratón Germinal

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

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Aislamiento de células madre de la retina del ojo del ratón

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Aislamiento de células de la glándula salival del ratón madre

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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

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

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

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

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

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...