Este trabalho foi financiado pelo National Institutes of prêmio Saúde R01 HL105734 para VVPMDL é suportado, em parte, pelo National Institutes of Health Research Career Award K08 HL094748. VVP é um pesquisador inovador da American Heart Association apoiada pela concessão 11IRG900384.

1 Preparar as soluções necessárias e Equipamentos <ol><li> Primeiro, esterilizar os instrumentos cirúrgicos (pinças retas forma, fórceps, tesouras forma curva de forma reta, e tesouras forma curva) em autoclave por 15 min. a 121 ° C. Preparar dois conjuntos de instrumentos, onde um conjunto é usado para dissecar os corações e o outro conjunto irá ser utilizados para triturar o tecido. </li><li> Dissolve-se um pacote de meios de cultura MCDB153 em 900 ml de esterilizado, água filtrada e agita-se lentamente até que o pó esteja dissolvido. Adicionar 1,2 g de bicarbonato de sódio, ou de 15,7 ml de uma solução de bicarbonato de sódio [7,5% w / v], por cada litro de volume final de meio que está sendo preparada e agita-se até se dissolver. Ajustar o pH para 7,2 por adição de 1 N ou HCl ou NaOH 1 N, em seguida verter os meios de comunicação através de um filtro de topo de 0,2 um frasco sob condições estéreis num capuz de cultura de tecidos (cabine de segurança biológica). Em seguida, coletar o material e armazene-o a 4 ° C. </li><li> Prepare os suplementos paraadicionar ao meio de cultura (Lista de Materiais). Os suplementos devem ser adicionados ao meio antes de apenas meios de comunicação é necessária para o plaqueamento das células. </li><li> Prepare DPBS com FBS a 10% mais antibióticos (penicilina e estreptomicina). </li><li> Em seguida, preparar uma solução de tripsina a 0,25% por adição de 0,25 g de tripsina (de pâncreas de bovino, actividade&gt; 2.500 unidades USP / mg, ultrapura; USB) a 100 ml de DPBS mais antibióticos (penicilina e estreptomicina). </li><li> Outros equipamentos necessários necessários para concluir este procedimento incluem um microscópio estereoscópico, placas de Petri (10 cm), tubos cônicos de 50 ml, filtros de células 40 mícrons, DPBS mais antibióticos, de 60 mm e placas de cultura de 35 mm placas de cultura. </li></ol> 2 Isolando Corações de Neonatais rato filhotes de cachorro <ol><li> Em primeiro lugar, obter P0 para p2 de rato neonatal filhotes (n = 6 a 8). Recomenda-se usar os filhotes gerados por meio de cruzamento DCT-Cre fêmeas homozigotas com os machos que são heterozigotos para tanto o R26R: alelo EYFP ou o alelo Z / EG para rotular omelanócitos semelhante. Ambos R26R: EYFP e Z / EG ratinhos estão disponíveis a partir de Jackson Laboratories (números 006148 e 003920 banco, respectivamente). </li><li> Em seguida, a eutanásia os filhotes recém-nascidos de rato, limpe o peito com um algodão embebido em álcool e em seguida, coloque os filhotes em uma placa de Petri de 10 cm com DPBS. </li><li> Cortar a pele sobre o peito e esterno com pinça e tesouras esterilizadas e em seguida, abra o baú com cuidado sob microscópio estereoscópico. Os corações dos filhotes recém-nascidos de rato são muito pequenos por isso tome cuidado para se certificar de remover todo o coração com os átrios anexados. </li><li> Lave os corações em DPBS e transferi-los para um novo prato de 10 cm Petri. </li></ol> 3. digestão enzimática <ol><li> Traga a placa de Petri com o coração excisadas para uma capa de cultura de tecidos (cabine de segurança biológica) e, em seguida, siga os passos abaixo usando técnicas estéreis na capa. </li><li> Em primeiro lugar, os corações lavar três vezes em DPBS estéril mais antibióticos. </li><li> Colocar os corações (n = 6-8) numa60 mm de placa de cultura e adicionar 6-8 mL de solução de tripsina a 0,5% para o prato. </li><li> Em seguida, o coração cortado em pedaços pequenos (menos de 1 mm de tamanho) e incubar a 37 ° C durante 30 min numa atmosfera de CO2 a 5%. </li><li> Após o período de incubação estiver completa, despejar a solução resultante de tecido cardíaco e tripsina a partir do prato para um tubo de 50 ml estéril cónica. </li><li> Em seguida, usando uma pipeta estéril de 10 ml, rapidamente mas suavemente, tritura-se a solução no tubo de 50 mL cónico 30-50 vezes. </li><li> Filtra-se a solução resultante através de um filtro de células de 40 mícrons no início de um novo tubo cónico limpo de 50 ml e recolher o filtrado. </li><li> Em seguida, o tubo de centrifugação de 50 ml a 240 x g numa centrífuga de bancada durante 5 minutos a temperatura ambiente. Em seguida, aspirar cuidadosamente o sobrenadante e ressuspender o sedimento restante em 6-8 ml de DPBS estéril. Repita este passo mais duas vezes. Esta etapa pelotas miócitos atriais e melanócitos cardíacos, mas não derrubar a fifibroblastos desde os fibroblastos são menores e permanecerão no sobrenadante. </li></ol> 4. chapeamento Cardiac Melanócitos como células <ol><li> Após a terceira lavagem, após a centrifugação, com cuidado drenar ou aspirar para fora da solução de lavagem (DPBS) e ressuspender o sedimento em 6 ml de meio de cultura MCDB aos quais foram adicionados os suplementos. </li><li> Agora, extrair a solução ressuspenso para uma pipeta de transferência estéril e colocar as células em uma placa de 35 mm, ou de um poço de uma placa de 6 poços, e reunir as células cardíacas isoladas a partir de três ratinhos neonatais em um prato de 35 mm ou bem. </li><li> Incubar as placas durante a noite a 37 ° C numa atmosfera de CO2 a 5%, aspirar fora do meio de cultura, juntamente com quaisquer células não ligadas e depois enxaguar as placas uma vez com DPBS. Em seguida, adicione meio fresco para as placas e substituir os meios de comunicação a cada 2 dias. As células isoladas a partir de corações neonatais ou embrionários pode ser mantido em cultura durante até 3 semanas, enquanto que as células isoladas de ouvir adultots podem ser mantidas em cultura durante até 10 dias. </li></ol> Células 5 Isolando Cardiac Melanócitos como de rato adulto Coração <ol><li> Em primeiro lugar, retirar os corações de ratos maduros (3-4 semanas de idade, n = 3), através de uma esternotomia linha média após a administração de heparina 100 U intraperitoneal para os ratinhos e, em seguida, eles eutanásia. Recomenda-se usar pentobarbital para sacrificá camundongos para este procedimento, mas este medicamento não está disponível em todos os locais. </li><li> Em seguida, lave os corações excisadas em DPBS estéreis contendo antibióticos em uma capa de cultura de tecidos (cabine de segurança biológica). Aperte os corações um pouco com uma pinça de dissecação curvas para remover qualquer vestígio de sangue restante deles e, em seguida, enxaguar os corações em DPBS mais uma vez. </li><li> Remover os átrios e anel atrioventricular dos corações (estas estruturas contêm as células de melanócito-like) e coloque as amostras de tecido excisadas em um prato de 35 mm. </li><li> Corte os corações em pedaços pequenos usando uma tesoura curva e forçaps e, em seguida, adicionar 6-8 mL de solução de tripsina a 0,5% para o prato. Em seguida, os pratos incubar a 37 ° C durante 45-60 min. </li><li> Agora, siga os passos do procedimento acima a partir do passo 3.6 em diante. </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>

Não temos nada a divulgar.

Melanócitos cardíacas estão presentes em locais anatômicos dentro do coração que normalmente dão origem a gatilhos arritmia atrial. Essas regiões incluem as veias pulmonares, o anel atrioventricular, a posterior do átrio esquerdo e do forame oval (Figura 4). Para compreender o papel dessas células desempenham na arritmogênese, desenvolvemos técnicas para isolá-los e estudar sua fisiologia. No entanto, reconhecemos que melhorar esta técnica para produzir quantidades mais saudáveis ​​e ...

Nós recentemente identificada uma população de células romance no coração de camundongos e humanos que expressam enzimas melanina-síntese e morfologicamente se assemelham melanócitos cutâneos. Chamamos &#39;melanócitos cardíacos&#39; essas células e descobrimos que eles estão presentes em locais anatômicos a partir do qual arritmia atrial desencadeia muitas vezes se originam em seres humanos. Encontramos também melanócitos cardíacos contribuir para arritmias atriais em camundongos com deleção da linhagem germinativa de Dct. Melanócitos cardíacas são escassamente distribuídos por todo o átrio do rato onde eles compreendem menos de 0,1% de todas as células atriais. Para investigar as propriedades elétricas e biológicos de melanócitos cardíacos foi desenvolvido um procedimento para isolá-los do coração mouse usando marcadores específicos de melanócitos Cre-induzida. Nosso procedimento inicial foi desenvolvido daqueles usados ​​para isolar cardiomiócitos murinos neonatais e rendeu um pequeno número de células adequadas para gravações de patch clamp ou análise de PCR. No entanto, para melhorar a curarº e viabilidade de melanócitos cardíacas, para mais aprofundada análise eletrofisiológica ou estudos bioquímicos, foi desenvolvido um procedimento refinado e as destinadas a isolar melanócitos cutâneos. O processo descrito e demonstrado neste teste tem a vantagem de produzir melanócitos cardíacas saudáveis, que podem ser plaqueadas como uma população pura ou com cardiomiócitos.

<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

Foi descoberta uma nova população de células de melanócitos tipo (também conhecido como melanócitos cardíaca) nos corações de ratos e seres humanos, que contribuem para os gatilhos de arritmias atriais em ratinhos. Para investigar as propriedades elétricas e biológicos de melanócitos cardíacos foi desenvolvido um procedimento para isolá-los do rato corações que nós derivados das destinadas a isolar cardiomiócitos murinos neonatais. A fim de obter melanócitos cardíacos saudáveis ​​adequados para mais extensa patch clamp ou estudos bioquímicos, foi desenvolvido um procedimento refinado para isolar e cultivar melanócitos cardíacos baseados naqueles originalmente projetado para isolar os melanócitos cutâneos. O procedimento refinado é demonstrado nesta revisão e produz um maior número de células de melanócito-like saudáveis ​​que pode ser banhado em uma população pura ou com cardiomiócitos.

O protocolo descrito neste artigo é uma técnica aprimorada em comparação com o que anteriormente publicado para isolar melanócitos cardíacos do coração murino. Este procedimento emprega a utilização de marcadores fluorescentes accionados por síntese de melanina-específica enzima Cre-recombinase para marcar as células de melanócitos, como cardíacas. É importante a utilização de um marcador para rotular melanócitos cardíacas quando se trabalha com células embrionárias ou neonatais recém-isolados, um...

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

Neste protocolo, w e identificou um novo população de células de melanócito-like (também conhecido como melanócitos cardíacos) nos corações de ratos e humanos que contribuem para a arritmia atrial dispara em camundongos.

Isolar células de melanócito-like primárias do coração 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 visualizações.  University of Pennsylvania.  Neste protocolo, w e identificou um novo população de células de melanócito-like (também conhecido como melanócitos cardíacos) nos corações de ratos e humanos que contribuem para a arritmia atrial dispara em camundongos. 

Vídeo: 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.

Isolar células-tronco de tecidos moles músculo-esqueléticas

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

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.

Detecção de Óxido Nítrico e ânion radical superóxido por espectroscopia de ressonância paramagnética eletrônica a partir de células usando armadilhas para spins

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.

Seleção e isolamento de colônias de células-tronco pluripotentes induzidas a partir de fibroblastos adultos reprogramado

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 de células imunes de tumores primários

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.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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.

Isolamento de proteínas a partir da Embryonic Rato Coração Região Válvula Desenvolvimento

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 cultura primária de células de camundongo aórtica endoteliais

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-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

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.

Culturas de células primárias do Mouse epithelium Retinal do Pigment

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, caracterização e diferenciação de células-tronco cardíacas do coração de rato 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 ...