Gostaríamos de agradecer a David R. Rodriguez, MA para o design gráfico e fotografia neste manuscrito. Este trabalho foi apoiado pelo financiamento do GCCRI, o NIH / NIA (5R21AG033339) eo Apoio Grant Cancer Center (P30CA054174) à facilidade UTHSCSA Citometria de Fluxo Core e da Facilidade UTHSCSA avançada Ácidos Nucleicos Core.

Os autores declaram não haver interesses financeiros concorrentes.

O ensaio de mutagénese in vivo aqui descrita baseia-se no modelo de ratinho transgénico LacI originalmente gerado por 18 Kohler et al. Este modelo utiliza um vector de fago λ portador de um gene repórter de lacl. Os dois sítios cos do vector que flanqueiam permitir uma recuperação relativamente simples e subsequente empacotamento em partículas de fago infecciosas, utilizados para infectar E. coli. Uma placa azul será gerado por infectadas com fago...

Na maioria dos tecidos, células diferenciadas têm uma duração de vida limitada. Para manter a integridade funcional, células estaminais de tecidos específicos de longa duração produzir continuamente células progenitoras que, por sua vez dão origem às células totalmente diferenciadas requeridas para a função daquele tecido particular. As células-tronco também reabastecer seu próprio compartimento através de um processo chamado de auto-renovação. Assim, as células-tronco são responsáveis ​​por manter a integridade funcional do tecido eles residem dentro, portanto, é imperativo que eles são equipados com mecanismos robustos para detectar e potencialmente reparar o DNA danificado. Se não, eles podem adquirir múltiplas perturbações genômicos (potencialmente perigosos), que podem ser herdadas por seus descendentes. A compreensão de como as células-tronco salvaguardar seu genoma durante o tempo de vida de um organismo é uma questão importante e pode nos ajudar a entender por que a instabilidade genômica está relacionada com o câncer e outras doenças relacionadas com a idade (revisada em 1,2). <p class = &quot;jove_content&quot;&gt; Controlando integridade genómica de um tecido ao nível das células estaminais ou de populações de células progenitoras no início pode ser conseguida quer eliminando haste com defeito (ou progenitora) por meio de células de morte celular, senescência ou diferenciação e / ou por reparação eficiente ADN de danificado. Estudos recentes demonstraram que é possível medir a certos tipos de reparação do ADN directamente nestas populações raras 3-6. Verificou-se que, por exemplo, no sistema hematopoiético, quebras duplas ADN cadeia pode ser reparada por recombinação homóloga (HR) ou a extremidade não-homóloga de união (NHEJ), sendo este último um processo de reparação de baixa fidelidade e, portanto, o aumento do risco de fazer erros. Ambos estão sendo utilizados em células estaminais hematopoiéticas (HSCs) de 4,5, no entanto, em murganhos, parece que é predominantemente NHEJ no HSCs enquanto que as células progenitoras iniciais utilizam RH 4. Uma observação semelhante foi feita por células-tronco na pele 6. Curiosamente, em humano HSCs HR, não NHEJ,parece ser o mecanismo de reparo de escolha para a vertente breaks duplos 3. Se essa diferença funcional entre as duas espécies é real ou apenas representa uma diferença tecnológica ou experimental continua a ser visto. Um repertório de células-tronco para reparar o DNA danificado é susceptível de incluir outros mecanismos de reparo do DNA, como a reparação por excisão de bases (BER), reparo por excisão de nucleotídeos (NER) e reparação incompatibilidade (MMR). BER e NER são responsáveis ​​pela reparação de lesões únicas ou múltiplas pares de bases no DNA fita simples, enquanto descasamentos correções MMR base-base e alças de inserção / exclusão; estes tipos de danos no DNA não pode ser reparado por NHEJ ou RH. Apoiando esta noção de diversos estudos do sistema hematopoiético que indicam uma ligação entre as alterações em uma destas vias e anormalidades no compartimento HSC 7-9, bem como um risco aumentado de desenvolvimento de síndrome mielodisplásica 10-16, uma doença que se origina naHSC e que está associada com o aumento da instabilidade genómica como a doença progride 17. Até o momento, não foram relatados medidas de BER, NER e MMR diretamente em HSCs. Além de elucidar os vários processos que controlam a integridade do tecido a um nível mecanístico, é imperativo para ser capaz de medir a extensão de ADN mutado, de modo que as consequências das aberrações em que um destes processos pode ser testada, por exemplo, normal versus geneticamente células-tronco modificadas ou no velho contra jovens. No entanto, o desenvolvimento de um ensaio relevante é difícil devido à escassez de células estaminais de tecidos específicos e a falta de condições de cultura que preserva &quot;stemness&quot;. Além disso, um tal ensaio deve ser modificável para manipulações genéticas e ambientais. Uma possível solução para estas limitações e exigências é a utilização de modelos de rato, que são especificamente projectados para detectar mutações no DNA. Mulmodelos de ratinhos transgénicos tiple para a detecção de mutações têm sido desenvolvidos. Por exemplo, o LACI ratinhos transgénicos 18 transportar um vector de fago λ recuperável que codifica o sistema LacI repórter, em que o gene codifica um supressor de LacI do operador Lac e serve como o repórter mutação. Após a mutação do gene LacI, o operador Lac é activado e β-galactosidase produzida. cliva β-galactosidase do substrato cromogénico X-gal (5-bromo-4-cloro-e-indolil-β-D-galactopiranósido), o que a torna azul. Os cos locais de acompanhamento do vetor LacI permite a recuperação fácil por proteínas do fago lambda e posterior infecção de E. coli. Após incubação de E. infectado coli em agarose que contém o substrato X-gal, as placas podem ser marcados. As placas azuis conter um mutante putativo Lac-I transportando fago, enquanto placas claras porto não mutantes. A freqüência de placas azuis (entre ªe as claras) indica que a frequência de mutação na população de células original é o DNA foi extraído a partir de. Além disso, o fago λ hospeda o alvo LacI pode ser facilmente sequenciada utilizando técnicas de PCR para análise relativamente alto rendimento. Seqüenciamento múltiplos genes mutantes LacI irá revelar informações importantes sobre o espectro de mutação, o que pode apontar para possíveis deficiências nas vias de reparo de DNA específicas ou eventos genotóxicos específicos. O sistema transgênico LacI foi padronizado em vários laboratórios 19 e os reagentes estão disponíveis comercialmente. Uma grande desvantagem do sistema LacI é a capacidade limitada para detectar grandes deleções ou rearranjos e, portanto, outros métodos, por exemplo, FISH multi-color em metáfase precisam ser usados ​​para complementar essa deficiência. Dentro do vector de fago λ do modelo do rato LacI, existe um gene muito menor, CII, Disponível para análise de mutação. O seu tamanho e ao facto de que os mutantes podem ser seleccionados torna este um ensaio de trabalho intensivo e mais barato menos 20 do que a análise do gene LacI. No entanto, o gene LacI está mais extensivamente estudado para a mutagénese 21 e a sensibilidade do gene de mutações foi bem caracterizada de modo a que existe um claro entendimento dos resíduos de aminoácidos que produzem uma resposta fenotípica sobre um substrato cromogênico 22-25. Outros modelos de ratinho para a detecção de mutações incluem a utilização do ΦX174 ou os transgenes LacZ. O modelo de camundongo transgênico ΦX174, com o Um original de: T → G: mutação reversão C ensaio 26 ou no ensaio de mutação para a frente 27, que permite a detecção de um espectro de substituição de pares de base, representa um sistema menos oneroso do que o modelo LacI. No entanto, a tela mutacional no ensaio para a frente não é trivial e a especificação mutaçãotro do transgene ΦX174 não é tão bem caracterizados como a do LACI. Em modelos de ratos portadores transgenes LacZ, o repórter mutacional LacZ é recuperado utilizando E. células hospedeiras de E. coli que são sensíveis à galactose e meio contendo galactose 28. Uma desvantagem deste sistema é que a recuperação do alvo LacZ também envolve a digestão com endonuclease de restrição seguida por ligação e a electroporação de E. coli hospeda, tornando assim difícil para adaptar o sistema para um pequeno número de células. Embora não seja uma exigência absoluta para o trabalho com populações de células tronco / progenitoras (sempre se pode começar com mais ratos), se um grande número de células são necessárias (por exemplo, milhões ou mais) ele vai rapidamente tornar-se impraticável e de custo proibitivo. Além disso, o tamanho relativamente grande de LacZ, enquanto proporciona um repórter sensível mutacional, é pesado e mais dispendioso para a análise de sequência de ADN e determminação de espectros de mutações. Uma grande vantagem deste modelo no entanto, é a sua capacidade de detectar grandes deleções e inserções, bem como os rearranjos cromossómicos. Uma vez que todas as células nos modelos de ratinho transgénicos LacI, LacZ ΦX174 e transportar o sistema repórter, qualquer destes modelos de rato pode ser utilizado para medir a mutagénese em qualquer tipo de células de interesse, incluindo as células estaminais e progenitoras, contanto que eles possam ser colhidos de forma fiável e em número suficiente. Porque nós tivemos uma vasta experiência com o modelo do rato LacI eo ensaio de mutação LacI, decidimos prosseguir este sistema ainda mais para a análise de mutagênese em tronco hematopoiéticas e populações progenitoras. O tecido hematopoiético é bem caracterizada em termos de fenótipo da superfície celular de seus componentes individuais, incluindo repovoamento de células-tronco de longo prazo, que são identificáveis ​​como a população extremamente rara de Lin - IL7R <sup&gt; - Sca-1 + c-kit + + (LSK) / Flk2 - CD150 + CD48 - células 29. . Mohrin et al 4 demonstraram que o ligeiramente maior população de LSK/Flk2 - células ainda são bons representantes para HSCs e significativamente diferente do mais primitivo progenitor mielóide comprometido população (CMP), quando se trata de estudar o reparo do DNA. Além disso, quando o LSK HSC enriquecido (Flk-2 + e Flk-2 -), as células foram comparados com o Lin - IL7R - Sca-1-c-kit + + (células progenitoras LS-K), há ainda uma diferença significativa nas NHEJ capacidade 5 entre o menos puro, caule enriquecida com células população LSK e as células progenitoras. Em nosso estudo utilizamos HSC-enriquecido LSK (Flk-2 + e Flk-2 -) células porque descobrimos que pelo menos 2 x 10 5 células são necessários para resultados consistentes e confiáveis ​​neste ensaio mutagênese, o número de células é extremamente difícilobter quando se classifica a LSK/Flk2 - população CD150 + CD48 ou mesmo o LSK/Flk2 - população (em termos de ratos, custos e praticidade). Este protocolo, com base no originalmente desenvolvida por Kohler et al. 18 descreve em pormenor a forma como a frequência de mutação espontânea de ADN pode ser determinada em células LSK e populações de células mielóides diferenciados, bem como células de medula óssea e do baço não separada definida.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<colgroup>
		<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

Nos últimos anos, tornou-se evidente que a instabilidade genômica é estreitamente relacionada com muitos transtornos do desenvolvimento, câncer e envelhecimento. Tendo em conta que as células-tronco são responsáveis ​​por garantir a homeostase e reparação tecidual ao longo da vida, é razoável supor que a população de células-tronco é fundamental para a preservação da integridade genômica dos tecidos. Portanto, o interesse significativo surgiu na avaliação do impacto de fatores endógenos e ambientais sobre a integridade genômica em células-tronco e seus descendentes, com o objetivo de compreender a etiologia das doenças com base em células-tronco. Ratinhos transgénicos LacI transportar um vector de fago λ recuperável que codifica o sistema LacI repórter, em que o gene LacI serve como o repórter mutação. O resultado de um gene mutante LacI é a produção de β-galactosidase que cliva um substrato cromogénico, transformando-azul. O sistema repórter LacI é i realizadon todas as células, incluindo as células tronco / progenitoras e podem ser facilmente recuperados e usados ​​para infectar posteriormente E. coli. Após incubação de E. infectado coli em agarose que contém o substrato correcta, as placas podem ser marcados; placas azuis indicam um gene mutante LacI, enquanto placas claras porto de tipo selvagem. A freqüência de azul (entre limpar) placas indica a frequência de mutantes na população célula original do DNA foi extraído. Sequenciação do gene mutante LacI vai mostrar a localização das mutações no gene e o tipo de mutação. O modelo de ratinho transgénico LacI está bem estabelecido como um ensaio de mutagénese in vivo. Além disso, os ratos e os reagentes para o ensaio estão disponíveis comercialmente. Aqui nós descrevemos em detalhes como este modelo pode ser adaptado para medir a freqüência de ocorrência espontânea mutantes de DNA em células-tronco enriquecido Lin - IL7R - Sca-1 + c-kit + + (LSCélulas K) e outras subpopulações de o sistema hematopoiético.

<html> O ensaio de mutagênese vivo mede um evento raro (PFUs mutante), entre muitos eventos (todos PFUs). Ao realizar o ensaio com pequeno número de células, é possível que o resultado é consideravelmente influenciado por resultados falsos positivos e falsos negativos. Para abordar esta questão foi realizado um experimento de diluição em série com células da medula óssea não fraccionada, colhidas a partir de três animais diferentes. Medimos a frequência de mutantes na medula óssea destes...

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

Descrevemos aqui um ensaio in vivo de mutagénese para pequenos números de células hematopoiéticas purificados utilizando o modelo de ratinho transgénico Lacl. O gene LacI pode ser isolado para determinar a frequência, a localização eo tipo de mutantes de DNA espontaneamente surgido ou após a exposição ao genotoxinas.

A identificação de DNA Mutações em Purificada-Tronco Hematopoéticas / células 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 visualizações.  UT Health Science Center at San Antonio.  Descrevemos aqui um ensaio in vivo de mutagénese para pequenos números de células hematopoiéticas purificados utilizando o modelo de ratinho transgénico Lacl. O gene LacI pode ser isolado para determinar a frequência, a localização eo tipo de mutantes de DNA espontaneamente surgido ou após a exposição ao genotoxinas. 

Vídeo: 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.

Identificação de genes desregulados Induzida pelo Herpesvirus sarcoma de Kaposi-associado (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 ...

Imunologia e Infecção

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.

Indução e Avaliação de recombinação mudança de classe em purificada Células B murino

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.

Formadoras de Colônias celular Assay (CFC) para células hematopoiéticas 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.

Sete Passos para Stellate Células

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.

Geração de células T específicas Multivirus para Prevenir / tratar infecções virais após transplante de células-tronco hematopoéticas alogênico

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.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago 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.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em 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.

Injeção sistêmica de células-tronco neurais / progenitoras em ratos com 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.

Visualizando interações DNA-proteína em células bacterianas ao vivo usando fotoativado única molécula de Rastreamento

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.

Avaliando o Desenvolvimento da murino células dendríticas plasmocitóides em placas de Peyer Usando transferência adoptiva de Progenitores hematopoiéticos

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