Questo lavoro è stato supportato dal National Institutes of award Salute R01 HL105734 a VVPMDL è supportato, in parte, dal National Institutes of Health Research Career Award K08 HL094748. VVP è un ricercatore innovativo della American Heart Association sostenuta da sovvenzioni 11IRG900384.

1 Preparare le soluzioni necessarie e attrezzature <ol><li> In primo luogo, sterilizzare gli strumenti chirurgici (pinze diritte forma, pinze forma curva, forbici forma diritta, e forbici forma curva) da loro autoclave per 15 min. a 121 ° C. Preparare 2 set di strumenti, in cui viene utilizzato un set per sezionare i cuori e l&#39;altro insieme saranno utilizzati per tritare il tessuto. </li><li> Sciogliere 1 bustina di MCDB153 cultura media in 900 ml di sterile, acqua filtrata e mescolare lentamente fino a che la polvere si è sciolta. Aggiungere 1,2 g di bicarbonato di sodio, o 15,7 ml di soluzione di bicarbonato di sodio [7,5% w / v], per ogni litro di volume finale di soluzione in fase di preparazione e mescolare fino alla dissoluzione. Regolare il pH a 7,2 aggiungendo o 1 N HCl o NaOH 1 N, quindi versare i media attraverso un filtro 0,2 micron top bottiglia in condizioni di sterilità in una cappa coltura tissutale (cabinet biosicurezza). Poi raccogliere i media e conservarlo a 4 ° C. </li><li> Preparare i supplementi aiaggiungere al terreno di coltura (Elenco materiali). Gli integratori devono essere aggiunti ai media poco prima è necessario il supporto per la placcatura le cellule. </li><li> Preparare DPBS con 10% FBS più antibiotici (penicillina e streptomicina). </li><li> Avanti, preparare una soluzione di tripsina 0,25% con l&#39;aggiunta di 0,25 g di tripsina (pancreas bovino, attività&gt; 2.500 unità USP / mg, ultrapura, USB) a 100 ml di DPBS più antibiotici (penicillina e streptomicina). </li><li> Altre apparecchiature necessarie necessaria per completare questa procedura includono uno stereomicroscopio, Petri-piatti (10 cm), tubi conici da 50 ml, filtri cellulari 40-micron, DPBS più antibiotici, 60 mm piatti della cultura e da 35 mm piatti della cultura. </li></ol> 2 Isolare Cuori da neonatali mouse Pups <ol><li> In primo luogo, ottenere P0 a P2 cuccioli di topo neonatale (n = 6 a 8). Si consiglia di utilizzare cuccioli generati da allevamento DCT-Cre femmine omozigoti con i maschi che sono eterozigoti per entrambi il R26R: EYFP allele o Z / EG allele per etichettare l&#39;cellule melanociti-like. Entrambi R26R: EYFP e Z / EG topi sono disponibili da Jackson Laboratories (numeri azionari 006.148 e 003.920 rispettivamente). </li><li> Avanti, eutanasia neonatale i cuccioli di topo, pulire il petto con un batuffolo imbevuto di alcool e poi mettere i cuccioli in un cm 10 Scatola di Petri con DPBS. </li><li> Tagliare la pelle sul petto e lo sterno con le pinze e forbici sterilizzate e poi aprire con cautela il petto sotto uno stereomicroscopio. I cuori di cuccioli neonati di topo sono molto piccole quindi fate attenzione a fare in modo di rimuovere l&#39;intero cuore con gli atri allegate. </li><li> Lavare i cuori in DPBS e trasferirli in un nuovo piatto di Petri di 10 cm. </li></ol> 3 Digestione enzimatica <ol><li> Portare la piastra di Petri con il cuore asportato ad una cappa di coltura tissutale (cabinet biosicurezza) e poi seguire le istruzioni riportate di seguito utilizzando tecniche sterili nella cappa. </li><li> In primo luogo, lavare i cuori per tre volte in DPBS sterili, più antibiotici. </li><li> Posizionare i cuori (n = 6-8) in un60-mm piatto di coltura e aggiungere 6-8 ml di soluzione di tripsina allo 0,5% al ​​piatto. </li><li> Quindi, tagliare i cuori in piccoli pezzi (meno di 1 mm di dimensioni) e incubare a 37 ° C per 30 minuti in una atmosfera di CO 2 5%. </li><li> Dopo il periodo di incubazione è completa, versare la soluzione risultante di tessuto cardiaco e tripsina dal piatto in un tubo conico da 50 ml sterile. </li><li> Poi, usando una pipetta sterile da 10 ml, rapidamente ma delicatamente, triturare la soluzione nel tubo conico da 50 ml 30-50 volte. </li><li> Filtrare la soluzione attraverso un filtro cella 40 micron sulla cima di una nuova pulita tubo conico da 50 ml, per raccogliere il filtrato. </li><li> Successivamente, centrifugare la provetta da 50 ml a 240 xg in una centrifuga da tavolo per 5 min a temperatura ambiente. Poi, delicatamente Aspirare il sopranatante e risospendere il pellet residuo in 6-8 ml di DPBS sterili. Ripetere questa operazione altre 2 volte. Questo passaggio Pellet miociti atriali e melanociti cardiache, ma non far cadere il fifibroblasti in quanto i fibroblasti sono più piccole e rimarranno nel surnatante. </li></ol> Celle 4 placcatura cardiaca melanocita-like <ol><li> Dopo il terzo risciacquo dopo la centrifugazione, scarico con attenzione o aspirare al largo della soluzione di lavaggio (DPBS) e risospendere il pellet in 6 ml di terreni di coltura MCDB a cui sono stati aggiunti i supplementi. </li><li> Ora, disegnare la soluzione risospeso in una pipetta di trasferimento sterile e posizionare le cellule in un piatto da 35 mm, o in un pozzetto di una piastra da 6 pozzetti, e in comune le cellule cardiache isolate da 3 topi neonati in un piatto da 35 mm o bene. </li><li> Incubare i piatti per una notte a 37 ° C in atmosfera di CO 2 5%, aspirare fuori dal terreno di coltura insieme a tutte le cellule non attaccati e poi sciacquare le piastre una volta con DPBS. Quindi aggiungere mezzi freschi per le piastre e sostituire i media ogni 2 giorni. Le cellule isolate da cuori neonatale o embrionali possono essere mantenuti in coltura per fino a 3 settimane, mentre le cellule isolate da sentire adultots possono essere mantenute in coltura per un massimo di 10 giorni. </li></ol> Cellule 5. isolamento Cardiac melanocita-come da Cuore topo adulto <ol><li> In primo luogo, rimuovere i cuori di topi maturo (3-4 settimane di età, n = 3) attraverso una sternotomia linea mediana dopo somministrazione di eparina 100 U intraperitoneale ai topi e poi li eutanasia. Si consiglia di utilizzare pentobarbital di eutanasia topi per questa procedura, ma questo farmaco non è disponibile in tutte le località. </li><li> Successivamente, lavare i cuori asportati in DPBS sterili contenenti antibiotici in una cappa coltura tissutale (cabinet biosicurezza). Spremere il cuore leggermente con dissettore curve per rimuovere il sangue residuo da loro e poi sciacquare i cuori in DPBS ancora una volta. </li><li> Rimuovere gli atri e anello atrioventricolare dal cuore (queste strutture contengono le cellule melanociti-like) e posizionare i campioni di tessuto asportato in un piatto da 35 mm. </li><li> Tagliare i cuori in piccoli pezzi con le forbici curve e forzaps e poi aggiungere 6-8 ml di soluzione di tripsina allo 0,5% al ​​piatto. Poi incubare le piastre a 37 ° C per 45-60 min. </li><li> Ora, seguire i passaggi della procedura di cui sopra a partire dal punto 3.6 in poi. </li></ol>

<ol>
	<li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

Non abbiamo nulla da rivelare.

Melanociti cardiache sono presenti in sedi anatomiche all&#39;interno del cuore che spesso danno luogo a trigger aritmie atriali. Queste regioni comprendono le vene polmonari, l&#39;anello atrioventricolare, posteriore sinistra dell&#39;atrio e del forame ovale (Figura 4). Per comprendere il ruolo di queste cellule svolgono in aritmogenesi, sviluppiamo tecniche per isolare loro e studiare la loro fisiologia. Tuttavia, abbiamo riconosciuto che il miglioramento di questa tecnica a cedere quantitativi più...

Abbiamo recentemente identificato una popolazione di cellule romanzo nel cuore di topi e gli esseri umani che esprimono enzimi melanina-sintesi e morfologicamente assomigliano melanociti cutanei. Abbiamo chiamato &#39;melanociti cardiaci&#39; queste cellule e l&#39;ho trovato sono presenti in posizioni anatomiche da cui aritmia atriale trigger spesso origine negli esseri umani. Abbiamo anche trovato melanociti cardiaci contribuiscono ad aritmie atriali nei topi con delezione linea germinale di Dct. Melanociti cardiache sono scarsamente distribuiti in tutto l&#39;atrio del mouse dove essi costituiscono meno dello 0,1% di tutte le cellule atriali. Per studiare le proprietà elettriche e biologiche dei melanociti cardiaci abbiamo sviluppato una procedura per isolarli dal cuore mouse utilizzando marcatori specifici melanociti Cre-inducibile. La nostra procedura iniziale è stato sviluppato da quelli utilizzati per isolare cardiomiociti murini neonatali e fruttate molto piccolo numero di cellule adatti per le registrazioni di patch clamp o analisi PCR. Tuttavia, per migliorare la guarigioneesima e la vitalità dei melanociti cardiaci, per più approfondita analisi elettrofisiologica o studi biochimici, abbiamo sviluppato una procedura raffinata da quelli progettati per isolare melanociti cutanei. La procedura descritta e dimostrata in questa recensione ha il vantaggio di produrre sani melanociti cardiaco che può essere placcato come una popolazione pura o con cardiomiociti.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>MCDB 153</td>
 <td>Sigma-Aldrich</td>
 <td>M7403</td>
 <td>Dissolve in: Water</td>
 </tr>
 <tr>
 <td>DPBS, no calcium no magnesium</td>
 <td>Invitrogen</td>
 <td>14190136 </td>
 <td>Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate, or Sodium Bicarbonate Solution, 7.5% </td>
 <td>Sigma-Aldrich </td>
 <td>S5761 or S8761 </td>
 <td>Water 
 Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140163 </td>
 <td>Stock Conc.: 100X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technology</td>
 <td>Certified</td>
 <td>Working Conc.: 4%</td>
 </tr>
 <tr>
 <td>TPA</td>
 <td>Sigma-Aldrich</td>
 <td>P1585</td>
 <td>DMSO 
 Stock Conc.: 3.2 &mu;M 
 Working Conc.: 32 nM</td>
 </tr>
 <tr>
 <td>Transferrin</td>
 <td>Sigma-Aldrich</td>
 <td>T8158</td>
 <td>DPBS 
 Stock Conc.: 0.1 mg/ml 
 Working Conc.: 1 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Dibutyryl cyclic AMP</td>
 <td>Sigma-Aldrich</td>
 <td>D0627</td>
 <td>DPBS 
 Stock Conc.: 0.5 mM 
 Working Conc.: 5 &mu;M</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>10 mM 
 acetic acid 
 Stock Conc.: 1 mg/ml 
 Working Conc.: 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Basic FGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Water 
 Stock Conc.: 4 ng/&mu;l 
 Working Conc.: 4 ng/ml</td>
 </tr>
 </tbody>
</table>

isolating primary melanocyte-like cells from the mouse heart

Abbiamo identificato una popolazione romanzo di cellule melanociti-simile (anche conosciuto come melanociti cardiaci) nel cuore dei topi e gli esseri umani che contribuiscono a trigger di aritmia atriale nei topi. Per studiare le proprietà elettriche e biologiche dei melanociti cardiaci abbiamo sviluppato una procedura per isolarli dai cuori di topo che derivati ​​da quelli progettati per isolare cardiomiociti murini neonatali. Al fine di ottenere melanociti cardiache sane adatte per una più ampia patch clamp o studi biochimici, abbiamo sviluppato una procedura raffinata per isolare e placcatura melanociti cardiaci basati su quelli originariamente progettato per isolare melanociti cutanei. La procedura raffinata è dimostrato in questa recensione e produce un numero maggiore di cellule melanociti-come sani che può essere placcato come una popolazione pura o con cardiomiociti.

Il protocollo descritto in questo articolo è una tecnica migliorata rispetto a quella che abbiamo già pubblicato per isolare melanociti cardiache dal cuore murino. Questa procedura impiega l&#39;uso di marcatori fluorescenti guidati da sintesi della melanina specifica-enzima Cre-ricombinasi di etichettare cellule melanociti-simile cardiaci. E &#39;importante utilizzare un marcatore per etichettare melanociti cardiaci quando si lavora con le cellule embrionali o neonatali appena isolate, dal momento che le differenze m...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

In questo protocollo, w e identificato una popolazione romanzo di cellule melanociti-simile (anche conosciuto come melanociti cardiaci) nel cuore dei topi e gli esseri umani che contribuiscono ad aritmia atriale provoca nei topi.

Isolamento di cellule melanociti-come primario dal Cuore del mouse

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

isolating-primary-melanocyte-like-cells-from-the-mouse-heart

Research

JoVE Journal

Biology

10.4K Views.  University of Pennsylvania.  In questo protocollo, w e identificato una popolazione romanzo di cellule melanociti-simile (anche conosciuto come melanociti cardiaci) nel cuore dei topi e gli esseri umani che contribuiscono ad aritmia atriale provoca nei topi. 

Video: Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Isolare cellule staminali da tessuti molli muscoloscheletrici

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Ricerca

Biologia

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Rilevamento di ossido nitrico e anione superossido radicale da spettroscopia di risonanza paramagnetica elettronica da cellule con Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selezione e isolamento di colonie di cellule pluripotenti indotte umane staminali riprogrammato da fibroblasti adulti

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

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

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

Isolamento di cellule immunitarie di tumori primari

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

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

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Colture cellulari primarie dall'epitelio retinico del pigmento del Mouse

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Methods Article

Isolamento di cellule melanociti-come primario dal Cuore del mouse