Nos gustaría dar las gracias a David R. Rodríguez, MA para el diseño gráfico y la fotografía en este manuscrito. Este trabajo fue apoyado por fondos del GCCRI, el NIH / NIA (5R21AG033339) y el programa de subsidios para el Centro de Cáncer (P30CA054174) a la instalación de UTHSCSA de Citometría de Flujo y el Fondo para UTHSCSA avanzada Nucleic Acids Core.

Los autores declaran no tener intereses financieros en competencia.

El ensayo in vivo de mutagénesis in descrito en el presente documento se basa en el modelo de ratón transgénico LacI originalmente generada por Kohler et al. 18 Este modelo utiliza un vector de fago λ que lleva un gen reportero lacI. Los dos sitios cos que flanquean el vector permite una recuperación relativamente simple y envasado posterior en partículas de fago infecciosas, utilizados para infectar E. coli. Una placa azul será generado po...

En la mayoría de los tejidos, las células diferenciadas tienen una vida útil limitada. Para mantener la integridad funcional,, las células madre específicas de tejido y de larga duración producen continuamente células progenitoras que a su vez dan lugar a las células completamente diferenciadas necesarios para la función de ese tejido particular. Las células madre también reponer su propio compartimento a través de un proceso llamado auto-renovación. Por lo tanto, las células madre son responsables de mantener la integridad funcional del tejido que residen pulg Por lo tanto, es imperativo que están equipadas con mecanismos robustos para detectar y potencialmente reparar el ADN dañado. Si no, se pueden adquirir múltiples perturbaciones genómicos (potencialmente nocivos), que pueden ser heredadas por sus descendientes. La comprensión de cómo las células madre salvaguardar su genoma durante el período de vida de un organismo es una pregunta importante y puede ayudar a entender por qué la inestabilidad genómica está vinculado con el cáncer y otras enfermedades relacionadas con la edad (revisado en 1,2). <p clculo = &quot;jove_content&quot;&gt; El control de la integridad genómica de un tejido en el nivel de células madre o poblaciones de células progenitoras tempranas se puede lograr ya sea por la eliminación de vástago defectuosa (o progenitor) células a través de la muerte celular, la senescencia o la diferenciación, y / o por la reparación eficiente ADN de dañado. Estudios recientes han demostrado que es posible medir ciertos tipos de reparación del ADN directamente en estas poblaciones raras 3-6. Se encontró que, por ejemplo, en el sistema hematopoyético, roturas de doble cadena de ADN se pueden reparar mediante recombinación homóloga (HR) o de extremos no homólogos (NHEJ), siendo este último un proceso de reparación de baja fidelidad y por lo tanto un mayor riesgo de hacer errores. Ambos están siendo utilizados en las células madre hematopoyéticas (HSC) de 4,5, sin embargo, en ratones parece que es predominantemente de NHEJ en HSCs mientras que las células progenitoras tempranas utilizan HR 4. Una observación similar se hizo para las células madre de la piel 6. Curiosamente, en las CMH humano HR, no NHEJ,parece ser el mecanismo de reparación de la opción para la línea se rompe dobles 3. Si esta diferencia funcional entre las dos especies es real o simplemente representa una diferencia tecnológico o experimental aún está por verse. Un repertorio de células madre para reparar el ADN dañado es probable que incluya otros mecanismos de reparación del ADN, como la reparación por escisión de base (BER), la reparación por escisión de nucleótidos (NER) y desajuste de reparación (MMR). BER y NER son responsables de la reparación de lesiones únicas o múltiples de pares de bases en el ADN de una sola hebra, mientras que los desajustes correcciones MMR de base-base y los bucles de inserción / deleción; este tipo de daños en el ADN no pueden ser reparados por NHEJ o HR. En apoyo de esta noción son varios estudios del sistema hematopoyético que demuestra una relación entre las alteraciones en una de estas vías y anormalidades en el compartimiento HSC 7-9, así como un mayor riesgo de desarrollar el síndrome mielodisplásico 10-16, una enfermedad que se origina en laHSC y que se asocia con el aumento de la inestabilidad genómica conforme avanza la enfermedad 17. Hasta el momento, no se han reportado mediciones de BER, TNE y MMR directamente en las CMH. Además de la aclaración de los diversos procesos que controlan la integridad del tejido a un nivel mecanicista, es imperativo que ser capaz de medir la extensión de ADN mutado, de modo que las consecuencias de las aberraciones en uno de estos procesos se pueden probar, por ejemplo, en normal frente genéticamente células madre genéticamente o en viejo y joven. Sin embargo, el desarrollo de un ensayo relevante es difícil debido a la escasez de células madre específicas de tejido y la falta de condiciones de cultivo que conserva &quot;stemness&quot;. Además, un ensayo de este tipo debe ser modificable a las manipulaciones ambientales y genéticos. Una posible solución a estas limitaciones y requisitos es el uso de modelos de ratón que se han diseñado específicamente para detectar mutaciones en el ADN. MulSe han desarrollado modelos de ratones transgénicos ples para la detección de mutaciones. Por ejemplo, LacI ratones transgénicos 18 llevar un vector de fago λ recuperable que codifica el sistema reportero de LacI, en el que el gen LacI codifica un supresor del operador lac y sirve como reportero de la mutación. Tras la mutación del gen LacI, el operador lac se activa y se produce β-galactosidasa. escinde β-galactosidasa del sustrato cromogénico X-gal (5-bromo-4-cloro-E-indolil-β-D-galactopiranósido), lo que la convierte azul. Los sitios cos que flanquean el vector LacI permite una fácil recuperación de proteínas del fago lambda y la posterior infección de E. coli. Después de la incubación de E. infectada coli en agarosa que contiene el sustrato X-gal, las placas se puede puntuar. Las placas azules contienen un mutante putativo fago Lac-Me realización, mientras que las placas claras albergan los no mutantes. La frecuencia de placas azules (entre ªe las claras) indica la frecuencia de mutantes en la población original de células se extrajo el ADN de. Por otra parte, el fago λ que aloja el objetivo LacI se puede secuenciar fácilmente usando técnicas de PCR para el análisis de relativamente alto rendimiento. La secuenciación de múltiples genes mutantes LacI revelará información importante sobre el espectro de mutaciones, que a su vez puede señalar posibles deficiencias en las vías específicas de reparación del ADN o para eventos genotóxicos específicos. El sistema transgénico LacI se ha estandarizado a través de múltiples laboratorios 19 y los reactivos están disponibles comercialmente. Una desventaja importante del sistema de LacI es la capacidad limitada para detectar grandes deleciones o reordenamientos, por lo tanto, otros métodos, por ejemplo, peces de múltiples colores en metafase para untar necesitan ser utilizado para complementar esta deficiencia. Dentro del vector de fago λ del modelo de ratón LacI, hay un gen mucho más pequeño, CII, Disponible para el análisis de mutaciones. Su tamaño y el hecho de que los mutantes se pueden seleccionar hacen de este un ensayo de mano de obra intensiva y más barato menos 20 que el análisis del gen LacI. Sin embargo, el gen LacI se estudia más ampliamente para la mutagénesis 21 y la sensibilidad del gen de mutaciones ha sido bien caracterizado de modo que haya una clara comprensión de los residuos de aminoácidos que generan una respuesta fenotípica en un sustrato cromogénico 22-25. Otros modelos de ratón para la detección de mutaciones incluyen el uso de la ΦX174 o los transgenes LacZ. El modelo de ratón transgénico ΦX174, con el original: T → G: mutación de reversión C de ensayo 26 o el ensayo de mutación hacia adelante 27 que permite la detección de un espectro de sustituciones de pares de bases, representa un sistema menos costoso que el modelo de LacI. Sin embargo, la pantalla de mutaciones en el ensayo directo no es trivial, y la especificación de la mutaciónTRUM del transgén ΦX174 no está tan bien caracteriza-como la de la LacI. En modelos de ratones que llevan transgenes LacZ, el reportero LacZ mutacional se recupera utilizando E. células huésped de E. coli que son sensibles a la galactosa y medio que contiene galactosa 28. Un inconveniente de este sistema es que la recuperación de la diana LacZ también implica digestión con endonucleasas de restricción seguido por ligamiento y la electroporación de E. coli acoge, lo que dificulta la adaptación del sistema para un pequeño número de células. Aunque no es un requisito absoluto para el trabajo con poblaciones de células madre / progenitoras (siempre se puede empezar con más ratones), si se requieren grandes cantidades de células (por ejemplo, millones o más) se convertirá rápidamente poco práctico y un costo prohibitivo. Además, el tamaño relativamente grande de LacZ, mientras que proporciona un reportero mutacional sensible, es engorroso y más costoso para el análisis de secuencia de ADN y determminación de los espectros de mutación. Una ventaja importante de este modelo sin embargo, es su capacidad para detectar grandes deleciones e inserciones, así como reordenamientos cromosómicos. Dado que todas las células en los modelos de ratones transgénicos LacI, LacZ ΦX174 y llevan el sistema reportero, cualquiera de estos modelos de ratón se puede utilizar para medir la mutagénesis en cualquier tipo de célula de interés, incluyendo las células madre y progenitoras, mientras que puedan ser cosechadas de forma fiable y en número suficiente. Porque tuvimos una gran experiencia con el modelo de ratón LacI y el ensayo de mutación LacI, hemos decidido llevar a cabo este sistema aún más para el análisis de mutagénesis en madre hematopoyéticas y las poblaciones progenitoras. El tejido hematopoyético es bien caracterizado en términos de fenotipo de la superficie celular de sus componentes individuales, incluyendo las células madre de repoblación a largo plazo, que son identificables como la población extremadamente rara de Lin - IL7R <sup&gt; - Sca-1 + cKit + + (TSI) / Flk2 - CD150 + CD48 - células 29. . Mohrin et al 4 demostraron que la población ligeramente más grande de LSK/Flk2 - células son todavía buenos representantes de las HSC y significativamente diferente de las más primitivas progenitoras mieloides comprometidos población (CMP) cuando se trata de estudiar la reparación del ADN. Por otra parte, cuando el TSI enriquecido-HSC (Flk-2 + y Flk-2 -) las células se compararon con los Lin - IL7R - Sca-1-cKit + + (células progenitoras LS-K), todavía había una diferencia significativa en NHEJ capacidad 5 entre el menos puro, tallo población TSI enriquecida con células y las células progenitoras. En nuestro estudio utilizamos HSC enriquecido LSK (Flk-2 Flk-2 + y -) de las células, ya que encontramos que se requieren al menos 2 x 10 5 células para obtener resultados consistentes y confiables en este ensayo de mutagénesis, lo que el número de células es extremadamente difícilobtener cuando uno ordena la LSK/Flk2 - población CD150 + CD48 o incluso el LSK/Flk2 - población (en términos de los ratones, los costos y practicidad). Este protocolo, basada en la que originalmente desarrollada por Kohler et al. 18 describe en detalle cómo la frecuencia de mutantes de ADN espontánea puede ser determinada en células LSK y poblaciones de células mieloides diferenciadas, así como células de la médula ósea y del bazo no separada definida.

<table border="0" cellpadding="0" cellspacing="0" width="691">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">LacI transgenic mice&#160;</td>
			<td style="width:245px;">BioReliance Corporation</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">720202</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 E. coli</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">200221</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA size standards &#8211; lambda ladder</td>
			<td>Bio Rad</td>
			<td>170-3635</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.025 mM pore size membranes&#160;</td>
			<td>Fisher (Millipore)</td>
			<td>VSWP 025 00</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">245 mm2 bioassay dishes (trays)&#160;</td>
			<td>Fisher (Corning)</td>
			<td>07-200-600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NZY broth (powder)</td>
			<td>Fisher (Teknova)</td>
			<td>N1144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Fisher</td>
			<td>E-3120-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N, N-dimethylformamide</td>
			<td>Fisher</td>
			<td>AC34843-5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal</td>
			<td>Research Products Intl Corp (RPI)</td>
			<td>B71800-10.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3-115-852</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Extender Taq Polimerase kit</td>
			<td>5 PRIME</td>
			<td>2200500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agencourt AMPure XP cleanup kit</td>
			<td>Beckman/coulter</td>
			<td>A63880</td>
		</tr>
	</tbody>
</table>

identifying dna mutations in purified hematopoietic stem/progenitor cells

En los últimos años, se ha hecho evidente que la inestabilidad genómica está estrechamente relacionada con muchos trastornos del desarrollo, cáncer y envejecimiento. Dado que las células madre son responsables de asegurar la homeostasis del tejido y la reparación durante toda la vida, es razonable plantear la hipótesis de que la población de células madre es crítico para la preservación de la integridad genómica de tejidos. Por lo tanto, un interés significativo se ha producido en la evaluación del impacto de los factores endógenos y ambientales sobre la integridad genómica en las células madre y su progenie, con el objetivo de comprender la etiología de las enfermedades basados ​​en células madre. Ratones transgénicos LacI llevan un vector de fago λ recuperable que codifica el sistema reportero de LacI, en el que el gen LacI sirve como reportero de la mutación. El resultado de un gen mutado LacI es la producción de β-galactosidasa que escinde un sustrato cromogénico, convirtiéndose azul. El sistema indicador LacI es i llevado an todas las células, incluyendo las células madre / progenitoras y pueden ser fácilmente recuperados y utilizados para infectar posteriormente E. coli. Después de la incubación de E. infectada coli en agarosa que contiene el sustrato correcto, las placas se puede marcar; placas azules indican un gen mutante LacI, mientras que las placas claras puerto de tipo salvaje. La frecuencia de azul (entre claras) placas indica la frecuencia de mutantes en la población original de células se extrajo el ADN de. La secuenciación del gen mutante LacI mostrará la ubicación de las mutaciones en el gen y el tipo de mutación. El modelo de ratón transgénico LacI es bien conocido por ser un ensayo in vivo de mutagénesis in. Por otra parte, los ratones y los reactivos para el ensayo están disponibles comercialmente. A continuación se describe en detalle la forma en este modelo puede ser adaptado para medir la frecuencia de mutantes de ADN que ocurre de forma espontánea en el tallo enriquecida con células Lin - IL7R - Sca-1 + cKit + + (LSCélulas K) y otras subpoblaciones del sistema hematopoyético.

<html> El ensayo in vivo de la mutagénesis in mide un evento raro (UFP mutante) entre muchos eventos (todo UFP). Al realizar el ensayo con pequeño número de células, es posible que el resultado está influido considerablemente por los resultados falsos positivos y falsos negativos. Para solucionar este problema se realiza un experimento de dilución en serie con las células de la médula ósea no fraccionadas, cosechado de tres animales diferentes. Se midió la frecuencia de mutantes en la médula ...

Watch this Scientific Journal Video about Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells at JoVE.com

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells | Immunology and Infection | Video

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Aquí se describe un ensayo in vivo mutagénesis por un pequeño número de células hematopoyéticas purificadas utilizando el modelo de ratón transgénico LacI. El gen LacI puede ser aislado para determinar la frecuencia, la ubicación y tipo de mutantes de ADN espontáneamente surgido o después de la exposición a genotoxinas.

La identificación de mutaciones del ADN en Purificada hematopoyéticas células madre / progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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12.9K vistas.  UT Health Science Center at San Antonio.  Aquí se describe un ensayo in vivo mutagénesis por un pequeño número de células hematopoyéticas purificadas utilizando el modelo de ratón transgénico LacI. El gen LacI puede ser aislado para determinar la frecuencia, la ubicación y tipo de mutantes de ADN espontáneamente surgido o después de la exposición a genotoxinas. 

Video: Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

La identificación de genes inducida por la desregulación de Sarcoma de Kaposi asociado al virus del herpes (HVSK)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Investigación

Inmunología e Infección

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Inducción y evaluación de la recombinación de cambio de clase en las células murinas purificada B

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

La formación de colonias de células (CFC) para el ensayo de células hematopoyéticas humanas

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Establecimiento de un<em&gt; In vitro</em&gt; Sistema para estudiar el comportamiento intracelular de<em&gt; Candida glabrata</em&gt; En humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Inyección sistémica de Neuronales células madre / progenitoras en ratones con EAE crónica

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualización de la proteína-DNA en células bacterianas en directo mediante fotoactivado Tracking Single-molécula

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...