Vorremmo ringraziare David R. Rodriguez, MA per la progettazione grafica e la fotografia in questo manoscritto. Questo lavoro è stato sostenuto da un finanziamento della GCCRI, il NIH / NIA (5R21AG033339) e il Cancer Center Support Grant (P30CA054174) per l&#39;impianto UTHSCSA Citometria a flusso Core e il Fondo UTHSCSA avanzata acidi nucleici Core.

Gli autori dichiarano interessi finanziari concorrenti.

Il test in vivo mutagenesi qui descritta si basa sul modello di topo transgenico LacI originariamente generato da Kohler et al. 18 Questo modello utilizza un vettore fagico λ trasportano un gene reporter lacI. I due siti cos fiancheggianti il vettore consentire un recupero relativamente semplice e successivo confezionamento in particelle fagiche infettive, utilizzati per infettare E. coli. Una targa blu sarà generato da fago-infetti E. coli che ...

Nella maggior parte dei tessuti, cellule differenziate hanno una durata limitata. Per mantenere l&#39;integrità funzionale, le cellule staminali tessuto-specifiche a vita lunga continuamente producono cellule progenitrici che a loro volta danno origine alle cellule completamente differenziate necessari per la funzione di quel particolare tessuto. Le cellule staminali anche ricostituire il proprio scompartimento attraverso un processo chiamato auto-rinnovamento. Pertanto, le cellule staminali sono responsabili del mantenimento dell&#39;integrità funzionale del tessuto in cui risiedono in Pertanto, è imperativo che essi sono dotati di meccanismi solidi di percepire e potenzialmente riparare il DNA danneggiato. In caso contrario, essi possono acquisire più perturbazioni (potenzialmente dannosi) genomiche, che possono essere ereditate dalla loro progenie. Capire come le cellule staminali salvaguardare il loro genoma durante la vita di un organismo è una questione importante e può aiutarci a capire perché l&#39;instabilità genomica è collegata con il cancro e altre malattie legate all&#39;età (recensiti 1,2). <p class = &quot;jove_content&quot;&gt; Controllo dell&#39;integrità genomica di un tessuto a livello delle cellule staminali o popolazioni di cellule progenitrici precoce può essere ottenuto sia eliminando stelo difettoso (o progenitore) cellule attraverso la morte cellulare, senescenza o differenziazione, e / o mediante riparazione efficiente DNA danneggiato. Recenti studi hanno dimostrato che è possibile misurare alcuni tipi di riparazione del DNA direttamente in queste rare popolazioni 3-6. Si è constatato che, per esempio nel sistema ematopoietico, doppie rotture del DNA possono essere riparati mediante ricombinazione omologa (HR) o non omologhe fine unione (NHEJ), quest&#39;ultimo è un processo di riparazione di bassa fedeltà e così aumentato rischio di fare errori. Entrambi vengono utilizzati nelle cellule staminali ematopoietiche (HSC) 4,5, tuttavia, in topi sembra che prevalentemente NHEJ in CSE mentre le cellule progenitrici primi utilizzano HR 4. Un&#39;osservazione analoga è stata fatta per le cellule staminali della pelle 6. È interessante notare che, in umana CSE HR, non NHEJ,sembra essere il meccanismo di riparazione di scelta per le pause doppio filamento 3. Se questa differenza funzionale tra le due specie è reale o semplicemente rappresenta una differenza tecnologico o sperimentale resta da vedere. Il repertorio di una cellula staminale per riparare il DNA danneggiato è probabile che includono altri meccanismi di riparazione del DNA, come riparazione per escissione di base (BER), riparazione per escissione del nucleotide (NER) e mismatch repair (MMR). BER e NER sono responsabili per la riparazione di lesioni singole o multiple di coppie di basi nel DNA a singolo filamento, mentre i disallineamenti correzioni MMR base-base e cicli di inserzione / delezione; questi tipi di danno al DNA non possono essere riparati da NHEJ o HR. A supporto di questa nozione sono diversi studi dal sistema ematopoietico dimostrare un legame fra alterazioni in uno di questi percorsi e anomalie nel vano HSC 7-9, così come un aumentato rischio di sviluppare la sindrome mielodisplastica 10-16, una malattia che ha origine nelHSC e che è associato con l&#39;aumento dell&#39;instabilità genomica come la malattia progredisce 17. Come ancora, non sono stati segnalati misure di BER, NER, e MMR direttamente in CSE. Oltre a chiarire i vari processi che controllano l&#39;integrità del tessuto ad un livello meccanicistico, è indispensabile per poter misurare l&#39;entità del DNA mutato, in modo che le conseguenze di aberrazioni in uno di questi processi possono essere testati, ad esempio in normali rispetto geneticamente cellule staminali ingegnerizzate o in vecchio contro giovane. Tuttavia, lo sviluppo di un saggio rilevante è difficile a causa della scarsità di cellule staminali tessuto-specifiche e la mancanza di condizioni di coltura che conservano &quot;stemness&quot;. Inoltre, tale analisi deve essere modificabili a manipolazioni ambientali e genetici. Una possibile soluzione a queste limitazioni e prescrizioni è l&#39;uso di modelli murini che specificamente sono progettati per rilevare mutazioni del DNA. Mulsono stati sviluppati teplici modelli di topi transgenici per il rilevamento mutazione. Ad esempio, LacI topi transgenici 18 portano un recuperabile vettore fago λ codificante il sistema giornalista LacI, in cui il gene codifica una LacI soppressore dell&#39;operatore Lac e serve come il reporter mutazione. Su mutazione del gene LacI, l&#39;operatore Lac è attivato e β-galattosidasi viene prodotto. β-galattosidasi scinde il substrato cromogenico X-gal (5-bromo-4-cloro-e-indolil-β-D-galattopiranoside) che trasforma blu. Le cos siti che fiancheggiano il vettore LacI consente un facile recupero da parte lambda proteine ​​fagi e la successiva infezione di E. coli. Dopo aver incubato infetto E. coli su agarosio contenente il substrato X-gal, placche possono essere assegnati. Targhe blu contengono un mutante putativo Lac-I trasportano fago, mentre placche evidenti porto non-mutanti. La frequenza di placche blu (tra the quelli chiari) indica la frequenza nella popolazione mutante cella originale il DNA è stato estratto da. Inoltre, il fago λ ospita il bersaglio LacI può essere facilmente sequenziato utilizzando tecniche di PCR per l&#39;analisi relativamente elevato rendimento. Sequenziamento geni multipli Laci mutanti rivelerà importanti informazioni sulla mutazione spettro, che a sua volta può indicare le eventuali carenze negli specifici percorsi di riparazione del DNA o per eventi specifici genotossici. Il sistema transgenico LacI è stato standardizzato su più laboratori 19 ei reagenti sono disponibili in commercio. Uno dei principali svantaggi del sistema LacI è la limitata capacità di rilevare grandi delezioni o riarrangiamenti, quindi, altri metodi, ad esempio FISH multi-colore su spread metafase devono essere utilizzati per completare questa carenza. All&#39;interno del vettore fago λ del modello murino LacI, c&#39;è un gene molto più piccolo, CII, Disponibili per l&#39;analisi di mutazione. Le sue dimensioni e il fatto che i mutanti possono essere selezionati rende questo un meno laborioso e più economico dosaggio 20 rispetto all&#39;analisi gene LacI. Tuttavia, il gene LacI è più ampiamente studiato per mutagenesi 21 e la sensibilità del gene per mutazioni è stata ben caratterizzata in modo che vi è una chiara comprensione dei residui amminoacidici che generano una risposta fenotipica su un substrato cromogenico 22-25. Altri modelli murini per la rilevazione di mutazione includono l&#39;uso della ΦX174 o dei transgeni LacZ. Il ΦX174 modello di topo transgenico, con l&#39;A originale: T → G: C mutazione reversione dosaggio 26 o il forward test di mutazione 27 che permette la rilevazione di uno spettro di sostituzioni di coppie di basi, rappresenta un sistema meno costoso rispetto al modello LacI. Comunque, la schermata mutazionale nel saggio in avanti non è banale e le specifiche mutazioneTrum del transgene ΦX174 non è come ben caratterizzata come quella del LacI. In modelli murini che trasportano transgeni LacZ, il giornalista mutazionale LacZ viene recuperato utilizzando E. cellule ospiti coli che sono sensibili al galattosio e terreno contenente galattosio 28. Un inconveniente di questo sistema è che il recupero del bersaglio LacZ coinvolge anche la digestione di restrizione endonucleasi seguita da ligazione ed elettroporazione di E. coli ospita, rendendo così difficile adattare il sistema per un piccolo numero di cellule. Anche se non è un requisito assoluto per lavorare con popolazioni di cellule staminali / progenitrici (si può sempre iniziare con più topi), se un gran numero di cellule sono necessari (ad es milioni o più) diventa subito poco pratico e un costo proibitivo. Inoltre, le dimensioni relativamente grandi di LacZ, fornendo un reporter mutazionale sensibile, è ingombrante e costosa per l&#39;analisi di sequenza del DNA e determminazione degli spettri mutazione. Uno dei principali vantaggi di questo modello tuttavia, è la sua capacità di rilevare grandi delezioni e inserzioni, nonché riarrangiamenti cromosomici. Poiché tutte le cellule in modelli murini transgenici LacI, ΦX174 e LacZ portano il sistema giornalista, uno di questi modelli mouse può essere utilizzato per misurare la mutagenesi in qualsiasi tipo di cellula di interesse, comprese le cellule staminali e progenitrici, fintanto che possono essere raccolte in modo affidabile e in numero sufficiente. Perché abbiamo avuto una vasta esperienza con il modello del mouse LacI e il test di mutazione LacI, abbiamo deciso di perseguire questo ulteriore sistema per l&#39;analisi di mutagenesi in staminali ematopoietiche e le popolazioni progenitrici. Il tessuto emopoietico è ben caratterizzato in termini di superficie delle cellule fenotipo dei suoi singoli componenti, tra cui ripopolamento cellule staminali-lungo termine, che sono identificabili come la popolazione estremamente rara di Lin - IL7R <sup&gt; - Sca-1 + CKIT + + (TSS) / Flk2 - CD150 + CD48 - 29 cellule. . Mohrin et al 4 hanno dimostrato che la popolazione leggermente più grande di LSK/Flk2 - le cellule sono ancora buoni rappresentanti per CSE e significativamente diversa da quella più primitiva impegnati progenitore mieloide (CMP) della popolazione quando si tratta di studiare la riparazione del DNA. Inoltre, quando la LSK HSC-arricchito (Flk-2 + e Flk-2 -) le cellule sono state confrontate con la Lin - IL7R - Sca-1-CKIT + + (cellule progenitrici LS-K), c&#39;era ancora una differenza significativa in NHEJ capacità di 5 tra i meno puro, staminali popolazione di TSS cellule arricchito e le cellule progenitrici. Nel nostro studio utilizziamo HSC-arricchito LSK (Flk-2 + e Flk-2 -), le cellule, perché abbiamo scoperto che almeno 2 x 10 5 cellule sono necessari per risultati coerenti e affidabili in questo test di mutagenesi, questo numero di cellulare è estremamente difficileavere quando si ordina la LSK/Flk2 - popolazione CD150 + CD48 o anche il LSK/Flk2 - della popolazione (in termini di topi, costi e praticità). Tale protocollo, basata su quella originariamente sviluppata da Kohler et al. 18 descrive in dettaglio come la frequenza DNA mutante spontaneo può essere determinata in cellule LSK e popolazioni di cellule mieloidi differenziate così come le cellule del midollo osseo non separato e milza definito.

<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

Negli ultimi anni, è diventato evidente che l&#39;instabilità genomica è strettamente legata a molti disturbi dello sviluppo, tumori e invecchiamento. Dato che le cellule staminali sono responsabili di assicurare l&#39;omeostasi dei tessuti e la riparazione per tutta la vita, è ragionevole ipotizzare che la popolazione di cellule staminali è fondamentale per preservare l&#39;integrità genomica dei tessuti. Pertanto, notevole interesse è sorto nel valutare l&#39;impatto di fattori endogeni ed ambientali sull&#39;integrità del genoma nelle cellule staminali e la loro progenie, al fine di comprendere l&#39;eziologia delle malattie a base di cellule staminali. Laci topi transgenici portano un recuperabile vettore fago λ codificante il sistema giornalista LacI, in cui il gene LacI serve come il reporter mutazione. Il risultato di un gene mutato LacI è la produzione di β-galattosidasi che un substrato cromogenico, trasformandolo blu. Il sistema giornalista LacI i è effettuatan tutte le cellule, comprese le cellule staminali / progenitrici e può essere facilmente recuperati e utilizzati per infettare successivamente E. coli. Dopo aver incubato infetto E. coli su agarosio contenente il substrato corretta, placche può essere ottenuto; placche blu indicano un gene mutante LacI, mentre placche evidenti porto wild-type. La frequenza di blu (tra chiari) placche indica la frequenza nella popolazione mutante cella originale il DNA è stato estratto da. Sequenziamento del gene mutante LacI mostrerà la posizione delle mutazioni nel gene e il tipo di mutazione. Il modello di topo transgenico LacI è ormai consolidata come un test in vivo mutagenesi. Inoltre, i topi ed i reagenti per il dosaggio sono disponibili in commercio. Qui si descrive in dettaglio come questo modello può essere adattato per misurare la frequenza di verificarsi spontaneamente mutanti di DNA in cellule staminali arricchita Lin - IL7R - Sca-1 + CKIT + + (LSK) cellule e altri sottopopolazioni del sistema ematopoietico.

<html> Il test in vivo mutagenesi misura un evento raro (mutante PFUs) tra i molti eventi (tutto PFUs). Eseguendo i test con piccolo numero di celle, è possibile che il risultato è notevolmente influenzata da risultati falsi positivi e falsi negativi. Per risolvere questo problema abbiamo effettuato un esperimento di diluizione seriale con cellule del midollo osseo non frazionata, raccolte dalle tre diversi animali. Abbiamo misurato la frequenza dei mutanti nel midollo osseo di questi animali con 1,4 x 10 <sup...

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

Qui si descrive in vivo mutagenesi saggio per un piccolo numero di cellule ematopoietiche purificate utilizzando il modello di topo transgenico LacI. Il gene LacI può essere isolato per determinare la frequenza, la posizione ed il tipo di mutanti DNA sorto spontaneamente o dopo l&#39;esposizione a genotossine.

L&#39;identificazione del DNA Mutazioni in depurata ematopoietiche cellule staminali / progenitrici

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 Views.  UT Health Science Center at San Antonio.  Qui si descrive in vivo mutagenesi saggio per un piccolo numero di cellule ematopoietiche purificate utilizzando il modello di topo transgenico LacI. Il gene LacI può essere isolato per determinare la frequenza, la posizione ed il tipo di mutanti DNA sorto spontaneamente o dopo l'esposizione a genotossine. 

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.

Identificare i geni disregolazione indotta da sarcoma di Kaposi associato 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 ...

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Immunologia e infezioni

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.

Induzione e Valutazione della ricombinazione Classe Switch in depurata cellule B murini

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.

Formanti colonia cellulare (CFC) Analisi per cellule emopoietiche

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.

Sette passi per Stellate Cellule

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.

Generazione di Multivirus-cellule T specifiche per prevenire / curare infezioni virali dopo trapianto allogenico di cellule staminali ematopoietiche

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 seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da 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.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

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.

Iniezione sistemica di cellule staminali neurali / progenitrici nei topi con EAE cronica

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.

Visualizzare le interazioni proteina-DNA in cellule vive batteriche Uso fotoattivati ​​singola molecola di monitoraggio

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.

Valutare lo sviluppo delle cellule murine plasmocitoidi dendritiche in placche del Peyer Utilizzando trasferimento adottivo di ematopoietiche Progenitori

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